Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.


Edited by Gerald Hart
Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomerinverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu 472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu 616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2glucosidase and is the first bacterial GH found in the GH65 family.
Glucose is the most abundant monosaccharide in nature, and its oligomers and polymers have various properties and physiological functions. For example, starch, which is a glucose polymer with α-1,4and α-1,6-linkages, serves as an energy storage material in plants, whereas glucose residues play an important role in protein quality control during the processing of eukaryotic N-glycans (1,2). Carbohydrate active enzymes (CAZymes) are involved in the biosynthesis and degradation of diverse carbohydrates including glucosides and are classified based on amino acid sequence homology: these include various families of glycoside hydrolases (GHs), glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and auxiliary activities that have been established and registered in the CAZy database (http://www.cazy.org/) (3)(4)(5). GHs are divided into the largest number of families in the CAZy database, with 171 families established as of August 2021 (several families are now considered obsolete). Some GH families have been further grouped into clans (GH-A to GH-R) on the basis of their structural similarity and catalytic mechanism (6). Although many enzymes with various substrate specificities have been reported in GH families, there is still an abundance of putative GHs with unknown functions.
Conversely, GH65 GHs have been found only in eukaryotes, and to date, only two activities have been reported. Acid trehalases, found in fungi, are active under acidic conditions, but their biochemical properties are not clear (29,30). Protein αglucosyl-1,2-β-galactosyl-L-hydroxylysine α-glucosidase (PGGHG) was found in Homo sapiens and Gallus gallus approximately 40 years ago and releases glucose from a disaccharide unit (2-O-glucopyranosyl galactopyranose, Glc-α1,2-Gal) attached to a hydroxylysine (Hyl) residue of collagen (31). The genes for PGGHGs were recently identified and classified as members of the GH65 family, and recombinant enzymes showed hydrolytic activity against Glc-α1,2-Gal-Hyl and type IV collagen (32). No crystal structure of GH65 GHs has yet been reported, and their catalytic mechanism is still unknown.
Flavobacterium johnsoniae is a Gram-negative soil bacterium whose genome has been sequenced, an analysis of which has revealed that it possesses several putative GHs to degrade polysaccharides (33). In this study, we report a GH65 enzyme found in this bacterium that has a low amino acid sequence identity with other reported GH65 enzymes. This enzyme is not a GP but showed α-1,2-glucosidase activity, and its crystal structure, reported here, is the first structure from the GH65 GHs. This report therefore provides structural insight into substrate specificity and the catalytic mechanism of the GH65 GHs.

Biochemical characterization of recombinant FjGH65A
F. johnsoniae possesses three genes for GH65 proteins, namely, Fjoh_1401, Fjoh_2641, and Fjoh_4428. A phylogenetic tree shows that Fjoh_2641 and Fjoh_4428 are included in a different clade from the bacterial GPs and eukaryotic GHs, whereas Fjoh_1401 belongs to the maltose phosphorylase clade (Fig. 2). Fjoh_4428 has lower than 33% sequence identity to known GH65 enzymes, including the GPs and GHs, and Fjoh_2641 lacks 288 amino acid residues at the N-terminal compared with Fjoh_4428. In this study, we therefore decided to characterize the function and structure of Fjoh_4428 (hereafter FjGH65A). FjGH65A without the N-terminal signal peptide was produced as a His 6 -tag-fused protein in Escherichia coli BL21 (DE3), and approximately 10 mg of purified FjGH65A was obtained from a cell lysate from a 500 ml culture. The theoretical molecular weight of FjGH65A was 76.4 kDa, which is consistent with the size of a single band for FjGH65A on an SDS-PAGE (Fig. S1A). Gel filtration chromatography showed that the molecular weight of FjGH65A was 433 ± 0.92 kDa, suggesting that this protein was a hexamer in solution (Fig. S1B). To determine the substrates of FjGH65A, we first examined its activity toward α-glucobiose because other known GH65 enzymes are active on α-glucosides. FjGH65A displayed activity against kojibiose and produced only glucose in the presence or absence of inorganic phosphate, but hydrolytic activity toward other α-glucobioses was not detected by TLC (Fig. 3). This result indicates that FjGH65A is not a GP but a GH specific for α-1,2glucosidic linkage. Using the glucose oxidase-peroxidase method, we detected fainter hydrolytic activity for nigerose (0.063 ± 0.006 unit mg −1 ) than kojibiose (33.9 ± 1.2 unit mg −1 ); no activity was detected for the other disaccharides. We also found that FjGH65A hydrolyzed longer kojioligosaccharides, from kojitriose to kojipentaose, but a kinetic analysis showed that k cat /K m values for longer kojioligosaccharides were lower than for kojibiose (Table 1). In addition, FjGH65A showed no activity toward p-nitrophenyl α-glucopyranoside, which is a general substrate of exo-acting α-glucoside hydrolases. The optimum pH and temperature of FjGH65A were 5.5 and 40 C, respectively (Fig. S2, A and B). The enzyme was stable (>80% residual activity) up to 50 C after 30 min incubation and in a pH range of 4.5 to 9.0 (Fig. S2, C and D).

Anomeric configuration of products
To explore the catalytic mechanism of FjGH65A, the initial products of FjGH65A hydrolysis against kojibiose and α-D-glucopyranosyl fluoride (α-GlcF) were analyzed via normal-phase HPLC. This system can separate α-glucose and β-glucose (see Experimental procedures); the retention times of α-glucose and β-glucose were 10.0 and 10.5 min, respectively (Fig. S3). When unhydrolyzed kojibiose (0 min) was applied to the HPLC, two peaks were detected at retention times of 22 and 23 min with an area ratio of 49% and 51%, respectively (Fig. 4A). Considering the ratio of α-kojibiose and β-kojibiose in aqueous solution (38), the 22 min peak and the 23 min peak likely corresponded to α-kojibiose and β-kojibiose, respectively. In comparison with the chromatograms of the 0 and 0.5 min reactions, a small peak at a retention time of 10.0 min and a large peak at a retention time of 10.5 min appeared, whereas the peaks at retention times of 22 and 23 min decreased. The amounts of glucose and kojibiose were determined from the peak areas in the chromatograms (Fig. 4B). From the slopes of these plots, FjGH65A digested α-kojibiose and β-kojibiose at a ratio of 1:0.8 and produces αglucose and β-glucose at a ratio of 1:2.2. This result suggests that FjGH65A hydrolyzes kojibiose via an anomer-inverting mechanism (Fig. 4C). However, because these products were derived from both the nonreducing end α-glucose and the reducing-end α/β-glucose of α/β-kojibiose, it was difficult to clearly distinguish between them. To further elucidate the reaction mechanism, α-GlcF (k cat = 56.1 ± 1.8 s −1 , K m = 3.1 ± 0.2 mM) was used as a substrate. Similarly, β-glucose initially accumulated in the 10 min reaction, whereas α-glucose was produced with a delay as the hydrolysis reaction progressed (Fig. 4D). This result strongly supported the hypothesis that FjGH65A is an α-glucoside hydrolase with an inverting mechanism (Fig. 4E).

Overall structure
The crystal structure of FjGH65A was solved using the single-wavelength anomalous dispersion method using a KAuCl 4 -soaked crystal because the molecular replacement method using the reported structures of GH65 GPs failed. We determined the structure of the enzyme in unliganded form and in complex with glucose at 1.54 and 1.40 Å resolutions, respectively (Table 2). Of all 681 amino acid residues, we were able to successfully model residues 23 to 681. The FjGH65A crystals belong to the space group C2 and contain three monomers (named MolA, MolB, and MolC) in the asymmetric unit. PISA (https://www.ebi.ac.uk/pdbe/pisa/) analysis showed that FjGH65A forms a "dimer of trimers" hexamer related by the crystallographic two-fold rotational axis (Fig. 5A). This result is consistent with the gel-filtration chromatography result described above. The total surface area of the hexamer is 123,460 Å 2 , whereas the buried interface area is 31,510 Å 2 . All GH65 enzymes of known structure were dimers, and the amino acid residues responsible for hexamer formation in FjGH65A were not conserved in any reported GH65 enzymes (27,28). The monomer of FjGH65A comprises four regions as follows: an N-terminal β-sandwich domain (N-domain, residues 23-258), a helical linker region (residues 259-294), an (α/α) 6 -barrel catalytic domain (residues 301-641), and a C-terminal β-sheet domain (C-domain, residues 295-300 and 642-681) (Fig. 5B). A structural similarity search was then performed using the Dali server (39). GH65 enzymes such as kojibiose GPs, a loop (residues 71-78) in the N-domain is shorter than the corresponding region (residues 62-79) in LbMP (27). The loop is located in the interface of the "dimer of trimers" and is suggested to be involved in the hexamer formation of the FjGH65A. The FjGH65A C-domain consists of five β-strands, which are fewer than those of the other GH65 GPs.

Active site of FjGH65A
During the refinement, the F o − F c electron density for βglucose molecules was found in the glucose-complex structure of FjGH65A. Each monomer of FjGH65A binds four β-glucose molecules, three of which (named Glc1, Glc2, and Glc3) was observed at the center of the catalytic domain (Figs. 5B and 6A), Structure of GH65 α-1,2-glucosidase whereas the other (Glc4) is bound to the linker region (Figs. 5B and S4). The following descriptions are based primarily on MolA. The B factors of Glc1, Glc2, Glc3, and Glc4 are 15.5, 14.7, 23.4, and 33.2 Å 2 , respectively. Glc1 is located at subsite −1 (subsite nomenclature is according to Davis et al. (40)) and interacts with the side chains of four amino acid residues (Trp 343 , Asp 344 , Lys 538 , and Gln 539 ) via hydrogen bond (Fig. 6B). Glc2 is located at subsite +1 and forms hydrogen bonds with Trp 391 , Glu 392 , Thr 407 , and Glu 472 . Glc3 at subsite +2 interacts with the side chain of Trp 473 via fewer hydrogen bonds and is partially exposed to the solvent (Fig. 6B). It is unclear how kojioligosaccharide substrates bind the active site, but the reducing-end of substrates is thought to protrude out into the solvent. These observations are suggested to be consistent with the fact that the activity against longer kojioligosaccharides is lower than that against kojibiose. Similarly, GH97 α-glucoside hydrolase SusB prefers maltotriose to longer maltooligosaccharides because of less interaction with the substrate at subsite +3, which is open to solvent (41). Among the hydroxy groups of Glc2, the O2 atom of Glc2 is the closest to the C1 atom of Glc1 (distance = 3.2 Å) (Fig. 6C). The superposition of the glucose-complex and CsKP in complex with kojibiose (PDB 3WIQ) demonstrates that the orientation of Glc2 is similar to that of the reducing-end glucose of kojibiose in CsKP. Among the hydroxy groups of Glc3, the O2 atom of Glc3 is the closest to the C1 atom of Glc2 (distance = 4.0 Å).
The active sites of CsKP in complex with kojibiose (PDB 3WIQ) and LbMP in native form (PDB 1H54) were superposed onto the FjGH65A active site. The amino acid residues interacting with Glc1, as well as the general acid Glu 472 , are conserved in both CsKP and LbMP, whereas the amino acid residues surrounding Glc2 (Trp 391 , Glu 392 , and Thr 407 in FjGH65A) are conserved in CsKP (which acts on the same substrate as FjGH65A) but not in LbMP (Fig. 7, A and B). The sequence alignment of FjGH65A and the reported eukaryotic GH65 GHs, including fungal acid trehalases and PGGHGs, indicate that the subsite −1 residues are completely conserved whereas the subsite +1 residues are different from each other (Fig. 7, B and C). All the GH65 GPs whose structure has yet been determined to possess a phosphate-binding site consisting of lysine, histidine, and two serine residues (27,28). In CsKP in complex with glucose and phosphate (PDB 3WIR), a phosphate molecule makes polar interactions with the two serines and is surrounded by histidine and lysine (Fig. 8A). In FjGH65A, the two serine residues are replaced by Pro 575 and Ala 576 , and the histidine and lysine residues are replaced by Phe 625 and Met 330 (Fig. 8, A and B). Instead, FjGH65A has Glu 616 in the sterically similar position as the phosphatebinding site in GH65 GPs. Glu 616 is located in a good position to act as a general base, and Glu 472 is conserved as a general acid among GH65 GHs and GPs. We constructed the mutants E472Q and E616Q, where each glutamic acid residue was substituted with glutamine; both the mutants lost activity (<0.1% of wild type) toward kojibiose. Based on the primary structure alignment, Glu 616 is completely conserved among the GH65 GHs (Fig. 8B), suggesting that Glu 616 acts as a general base on FjGH65A and is therefore essential for hydrolytic reactions.
Comparison with clan GH-L enzymes GH15 and GH65 share an anomer-inverting mechanism and an (α/α) 6 -barrel catalytic domain. Based on these similarities, they are classified into clan GH-L and have been thought to have a related evolutionary origin (27). The overall structure of FjGH65A was compared with that of GH15 glucoamylase TtGA in complex with acarbose (PDB 1LF9). FjGH65A and TtGA share an N-domain, a linker region, and an (α/α) 6 -barrel catalytic domain (Fig. 9, A and B). Figure 9C shows the superimposition of Cα atoms of catalytic residues and conserved residues in FjGH65A and TtGA. The general acid and base catalysts are located on the loops between the α5 and α6 helices and between the α11 and α12 helices of the (α/ α) 6 -barrel catalytic domain and are structurally conserved. In TtGA, Asp 344 is located at the α2 helix and contributes to the capture of nucleophilic water molecules via hydrogen bonding with the O6 of glucose bound to subsite −1 (42). Tyr 337 is located between the α1 and α2 helices of TtGA and forms a hydrogen bond with the general base Glu 636 and is highly conserved in GH15 (43). In the superposition, the side chains of these residues overlap each other; Glc1 in FjGH65A and the Structure of GH65 α-1,2-glucosidase cyclohexene of acarbose in TtGA also overlap at subsite −1 (Fig. 9C).
On this enzyme, subsite +2 is more spacious than subsites −1 and +1, which strictly recognize kojibiose, suggesting that oligosaccharides with α-linkages other than α-1,2 at the reducing end, such as G2G6G, are acceptable (Fig. 10). Therefore, it is likely that FjGH65A plays a role in the degradation of the oligosaccharides that are products resulting from the hydrolysis of α-1,2-branched dextran by peripheral gene products such as dextranase FjDex31A and/or a putative GH66 dextranase (52). FjGH65A has a signal peptide, indicating that the enzyme is periplasmic or extracellular. However, the metabolic pathway of α-1,2-branched dextran in F. johnsoniae must still be investigated further.
Although the enzymatic properties of GH65 GHs, including fungal acid trehalases and vertebrate PGGHGs, have been reported, the general base had not been identified, and the catalytic mechanism had not been investigated. The general base residue was predicted only by mutational analysis of human PGGHG (32). Using kojibiose and synthetic α-GlcF, we identified the hydrolytic mechanism of FjGH65A as an inverting mechanism, which to our knowledge is the first time this has been done for a GH65 GH. The crystal structure and mutational analysis of FjGH65A revealed that Glu472 and Glu616 are the catalytic acid and base, respectively, and support this mechanism. In addition, it is suggested that GH15 enzymes and GH65 GHs have common catalytic machinery with sterically conserved residues, including the catalytic acid and base residues. This reinforces the theory that GH15 and GH65 share a common ancestral protein. For example, GH130 contains inverting GPs and GHs active on β-mannosides and is the only inverting GH family where the structures of both GPs and GHs have been reported to date (59,60). In GH130 βmannoside phosphorylases, the basic amino acid residues that interact with inorganic phosphate are conserved (60,61). By contrast, GH130 β-mannosidases have two glutamic acid residues that are expected to be involved in the hydrolysis at the position corresponding to the phosphate-binding site in GH130 β-mannoside phosphorylases (59). In addition, the amino acid residues forming subsite −1 are conserved between GH130 β-mannoside phosphorylase and β-mannosidase Structure of GH65 α-1,2-glucosidase (59,61). These features resemble the relationship between FjGH65A and the GH65 GPs.
Recently, the Conserved Unique Peptide Patterns program, a program for the functional annotation and subgrouping of proteins based on a new peptide-based similarity assessment algorithm, has been created for CAZymes (62,63). GH65 is grouped by the Conserved Unique Peptide Patterns from GH65:1.1 to GH65.16.1. FjGH65A is among these and is classified as GH65:8.1, where GH65 proteins from Elizabethkingia and Bacteroides also belong. The glutamate residues proposed as the general acid and general base were conserved, and amino acid residues that form a phosphatebinding site are not present in GH65:8.1. Tryptophan and glutamate residues corresponding to Trp 391 and Glu 392 of FjGH65A, which are important for the recognition of kojibiose, were also conserved in GH65:8.1. Therefore, it may be the case that GH65 enzymes classified as GH65:8.1 may actually be α-1,2-glucosidases.
In conclusion, FjGH65A is the first bacterial GH65 GH and is a novel inverting α-1,2-glucosidase that specifically hydrolyzes kojibiose, unlike previously reported enzymes. We propose α-1,2-D-glucoside glucohydrolase as the systematic name and α-1,2-glucosidase as the short name for FjGH65A. Moreover, its crystal structure was determined as the first GH65 GHs and revealed the structural commonalities and differences involved in the catalysis and substrate recognition in GH65 GHs and GPs. Our results will help us to better understand the mechanisms of hydrolysis and substrate Structure of GH65 α-1,2-glucosidase specificity in GH65 enzymes-including eukaryotic GHs-and to predict the functions of uncharacterized enzymes within not only GH65, but also other GH families that contain GPs.

Recombinant protein production and purification
FjGH65A (GenBank ID, ABQ07432.1) was initially predicted to have no signal sequence using the SignalP server (www.cbs.dtu.dk/services/SignalP/) (64) although the sequence contains some hydrophobic amino acid residues at the N-terminus. We found another candidate for the start codon of FjGH65A 39-bp upstream, whose translation product was predicted to have a signal sequence of 23 amino acid residues, consistent with the sequence found in the RefSeq database (WP_044048041.1). The amino acid residues are numbered according to WP_044048041.1 in this article. The gene for FjGH65A (ABQ07432.1, residues 14-681) was amplified from F. johnsoniae NBRC 14942 (ATCC 17061, UW101) by colony-direct PCR using KOD FX Neo DNA polymerase (Toyobo) and a pair of primers, FjGH65AΔ13-NheI-F and FjGH65A-XhoI-R (Table S1). The amplified gene product was then ligated into a pET28a (+) vector (Merck Millipore) using the NheI and XhoI restriction sites. The resulting plasmid was used as a template for inverse PCR with a pair of primers, FjGH65A-F and FjGH65A-R (Table S1), to construct the expression plasmid of the N-terminally Histagged FjGH65A without the signal sequence (residues 24-681). Site-directed mutagenesis was performed via inverse PCR with the desired primers (Table S1) using the recombinant FjGH65A expression plasmid as a template. The sequences of the constructs were verified by DNA sequencing. E. coli BL21(DE3) cells harboring the expression plasmid were grown in LB (1% tryptone, 0.5% yeast extract, and 1% NaCl) medium supplemented with 50 μg/ml kanamycin (Merck Millipore) at 37 C until the absorbance reached 0.6 to 0.8. Isopropyl β-D-1-(Merck Millipore) was added at a final concentration of 0.1 mM, and the culture medium was incubated at 20 C for 24 h. After induction, the cells in 500 ml of the culture medium were collected, resuspended in 30 ml of 20 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and

Enzyme assays
When α-glucobiose was used as a substrate, the hydrolysis activity of FjGH65A was analyzed via TLC with the following reaction conditions: 10 mM substrate and 10 μg/ml (130 nM) purified FjGH65A at 30 C for 10 min. The reaction solution and authentic standards (glucose, maltooligosaccharides, and β-glucose-1-phosphate) were spotted on TLC aluminum sheet silica gel 60 F254 and developed with 1-butanol:ethanol:water = 5:5:2. To calculate the specific activity of FjGH65A against  Figure 8. The overlaid residues of TtGA and acarbose are shown in blue and yellow, respectively. GH, glycoside hydrolase; GH65, glycoside hydrolase family 65; TtGA, glucoamylase from Thermoanaerobacterium themosaccharolyticum.

Kinetics studies
The initial velocities of the hydrolytic reactions for kojioligosaccharides, G2G6G, and G2G2G6G were determined using the 50 mM sodium citrate buffer (pH 5.5) and at least four concentrations of substrates were used, that is, 0.1 to 2 mM kojibiose, 0.1 to 2 mM kojitriose, 0.2 to 3 mM kojitetraose, 0.2 to 5 mM kojipentaose, 0.2 to 2 mM G2G6G, and 0.2 to 5 mM G2G2G6G. The enzyme concentrations used were 1 μg/ml (13 nM) against kojibiose, kojitriose, kojitetraose, and G2G6G or 10 μg/ml (130 nM) against kojipentaose and G2G2G6G. The amount of liberated glucose was quantified via the glucose oxidase-peroxidase method by using a Glucose C-II Test Kit. The same procedure was performed three times for each reaction. The reaction kinetics parameters were determined via nonlinear regression analysis implemented by KaleidaGraph (Synergy Software).

Analysis of the anomeric form of the product
Anomers of the hydrolytic products of kojibiose and α-GlcF were analyzed via normal-phase HPLC. The enzymatic reaction was performed in a 50 mM sodium citrate buffer (pH 5.5) at 30 C containing 100 mM of each substrate and 100 μg/ml (1.3 μM) FjGH65A. The reactions were carried out for 30 s, 1 min, 2 min, 3 min, 4 min, 5 min, and 6 min when kojibiose was used as the substrate; the reactions were carried out for 10 min, 35 min, 60 min, and 180 min when α-GlcF was used as the substrate. The reaction mixtures were then applied to a TSK-GEL amide-80 column (4.6 × 250; Tosoh) immediately after incubation and were eluted with 80% (v/v) acetonitrile at a flow rate of 1.2 ml/min at 25 C. The reaction products were detected using a refractive index detector (RID-10A, Shimadzu). The retention times of α-glucose (Merck Millipore) and β-glucose (Tokyo Chemical Industry) were determined in the same manner.

Crystallization and structure determination
FjGH65A (30 mg/ml in 20 mM sodium citrate buffer, pH 6.0 and 150 mM NaCl) was crystallized at 20 C by the hanging drop vapor diffusion method; 1 μl of protein solution was mixed with an equal volume of the mother liquor consisting of 12% (w/v) PEG3350 (Hampton Research), 0.3 M ammonium citrate buffer (pH 7.0), and 10 mM tris (2-carboxyethyl) phosphine hydrochloride (Hampton Research). The crystals were cryoprotected with a reservoir solution supplemented with 20% (v/v) ethylene glycol or 30% (w/v) glucose and quickly frozen in liquid nitrogen. For phase determination, the crystals were soaked in a reservoir solution supplemented with 10 mM KAuCl 4 at 20 C for 16 h before cryoprotection. Diffraction data were collected at the NW12 A beamline (Photon Factory). The data were first processed via XDS (66) and then scaled using SCALA (67) as implemented in the CCP4 package (68). The initial phase was determined via single-wavelength anomalous dispersion using a single crystal soaked in KAuCl 4 and the phase determination program Phaser (69) on CCP4. The unliganded structure of FjGH65A and the complex structure with glucose were determined using the molecular replacement program MOLREP (70). Manual model building was performed using COOT (71), and refinement was performed using REFMAC5 (72) and Translation/ Libration/Screw Motion Determination (73). Molecular images were made using PyMOL (Schrödinger LLC). Structural similarity searches were performed using the Dali server (39). Table 2 summarizes the data collection and refinement statistics.

Sequence alignment and phylogenetics
The protein sequences were aligned using Clustal Omega (74), and the figures were generated using ESPript 3.0 (75). For phylogenetic analysis, the protein sequences were aligned using MUSCLE (76), and the resulting alignment was used for generating a phylogenetic tree via the maximum likelihood method using MEGA 7 (77).

Data availability
The atomic coordinates and structure factors have been deposited in the Worldwide Protein Data Bank (http://wwpdb. org/) under accession codes 7FE3 and 7FE4. All other data are contained within the article.
Supporting information-This article contains supporting information.