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Structural Insights into the Epimerization of β-1,4-Linked Oligosaccharides Catalyzed by Cellobiose 2-Epimerase, the Sole Enzyme Epimerizing Non-anomeric Hydroxyl Groups of Unmodified Sugars*

Open AccessPublished:December 20, 2013DOI:https://doi.org/10.1074/jbc.M113.531251
      Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of β1,4-mannobiose to 4-O-β-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the β5-β6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.

      Introduction

      Cellobiose 2-epimerase (CE)
      The abbreviations used are: CE
      cellobiose 2-epimerase
      Man-Glc
      4-O-β-d-mannosyl-d-glucose
      AGE
      N-acetylglucosamine 2-epimerase
      PGI
      phosphoglucose isomerases
      Glc-Man
      4-O-β-d-glucosyl-d-mannose.
      (EC 5.1.3.11), which was first identified in the ruminal anaerobic bacterium, Ruminococcus albus (
      • Tyler T.R.
      • Leatherwood J.M.
      Epimerization of disaccharides by enzyme preparations from Ruminococcus albus.
      ), catalyzes interconversion of the d-glucose residue into a d-mannose residue at the reducing end of β1,4-linked oligosaccharides, including β1,4-mannobiose, cellobiose, and lactose. Epimerases acting on carbohydrates and its derivatives can be divided into 25 groups (EC 5.1.3.-) depending on their catalytic reactions and substrate specificities. Most epimerases, converting the configuration of non-anomeric hydroxyl groups, act on modified active substrates harboring phosphate groups or nucleotide diphosphate groups. In contrast to most sugar epimerases, CE catalyzes the epimerization of unmodified sugars at the C2 position. Recently, the metabolic pathway of mannan involving CE was postulated for the CE-producing bacteria, R. albus, and Bacteroides fragilis (
      • Senoura T.
      • Ito S.
      • Taguchi H.
      • Higa M.
      • Hamada S.
      • Matsui H.
      • Ozawa T.
      • Jin S.
      • Watanabe J.
      • Wasaki J.
      • Ito S.
      New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase.
      ,
      • Kawahara R.
      • Saburi W.
      • Odaka R.
      • Taguchi H.
      • Ito S.
      • Mori H.
      • Matsui H.
      Metabolic mechanism of mannan in ruminal bacterium, Ruminococcuss albus, involving two mannoside phosphorylases and cellobiose 2-epimerase. Discovery of a new carbohydrate phosphorylase, β-1,4-mannooligosaccharide phosphorylase.
      ). During mannan metabolism, CE converts β1,4-mannobiose, produced by hydrolysis of mannan or phosphorolysis of β1,4-mannooligosaccharides catalyzed by β1,4-mannanase (EC 3.2.1.78) or β1,4-mannooligosaccharide phosphorylase (EC 2.4.1.-), respectively, into 4-O-β-d-mannosyl-d-glucose (Man-Glc). The resulting Man-Glc is consequently degraded into α-d-mannosyl phosphate and d-glucose by 4-O-β-d-mannosyl-d-glucose phosphorylase (EC 2.4.1.281). CE also converts lactose into the rare oligosaccharide, epilactose (4-O-β-d-galactosyl-d-mannnose) (
      • Ito S.
      • Taguchi H.
      • Hamada S.
      • Kawauchi S.
      • Ito H.
      • Senoura T.
      • Watanabe J.
      • Nishimukai M.
      • Ito S.
      • Matsui H.
      Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme.
      ), which is useful for medical applications as a prebiotic agent. Epilactose stimulates bifidobacteria growth in vivo, suppresses the conversion of primary bile acid into secondary bile acid (
      • Watanabe J.
      • Nishimukai M.
      • Taguchi H.
      • Senoura T.
      • Hamada S.
      • Matsui H.
      • Yamamoto T.
      • Wasaki J.
      • Hara H.
      • Ito S.
      Prebiotic properties of epilactose.
      ), and promotes absorption of some minerals (
      • Nishimukai M.
      • Watanabe J.
      • Taguchi H.
      • Senoura T.
      • Hamada S.
      • Matsui H.
      • Yamamoto T.
      • Wasaki J.
      • Hara H.
      • Ito S.
      Effects of epilactose on calcium absorption and serum lipid metabolism in rats.
      ,
      • Suzuki T.
      • Nishimukai M.
      • Takechi M.
      • Taguchi H.
      • Hamada S.
      • Yokota A.
      • Ito S.
      • Hara H.
      • Matsui H.
      The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.
      ). Elucidation of the amino acid sequence of CE from R. albus (RaCE) facilitated the identification of CEs in other bacteria (
      • Ojima T.
      • Saburi W.
      • Yamamoto T.
      • Mori H.
      • Matsui H.
      Identification and characterization of cellobiose 2-epimerases from various aerobes.
      ). Among the CEs identified to date, Rhodothermus marinus CE (RmCE) is highly stable at high temperatures, and converts lactose into epilactose with high efficiency in contrast to the other known CEs (
      • Ojima T.
      • Saburi W.
      • Sato H.
      • Yamamoto T.
      • Mori H.
      • Matsui H.
      Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.
      ), including RaCE. Therefore, RmCE is very attractive as an enzyme for production of epilactose.
      Amino acid sequences of CEs show weak similarity to N-acetylglucosamine 2-epimerases (EC 5.1.3.8, AGEs) and aldose-ketose isomerases YihS. Recently, we determined the structure of RaCE. The overall structure of RaCE adopts an (α/α)6 barrel similar to the catalytic domains of AGE and YihS, and therefore CE comprise the AGE superfamily together with AGE and YihS (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ). The catalytic mechanism of CE was discussed based on structural comparison and mutational analysis of RaCE (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ). In CE, two essential histidine residues, corresponding to the putative general acid/base catalysts of AGEs, and a third histidine residue located at the bottom of the substrate binding site are required for catalysis. Epimerization catalyzed by CE is likely to proceed with the pair of histidine residues (RaCE-His-243 and RaCE-His-374) as general acid/base catalysts. Moreover, it appears that the roles of these two histidine residues as general acid/base catalysts may be reversed when the reverse reaction takes place. The histidine residues corresponding to RaCE-His-243 and RaCE-His-374 are completely conserved in enzymes belonging to the AGE superfamily, supporting the importance of these residues. These histidines were found in Anabaena sp. AGE (aAGE-His-239 and aAGE-His-382) (
      • Lee Y.C.
      • Wu H.M.
      • Chang Y.N.
      • Wang W.C.
      • Hsu W.H.
      The central cavity from the (α/α)6 barrel structure of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase contains two key residues for reversible conservation.
      ) and Salmonella enterica YihS (SeYihS-His-248 and SeYihS-His-383) (
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ). In contrast, the function of the third histidine in RaCE (RaCE-His-184) conserved in CE and YihS was proposed (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ). In the case of CE and YihS, the H2 proton of the unmodified sugars at the reducing end of substrates is not easily abstracted due to its high pKa value. Therefore, the third histidine facilitates deprotonation of the C2 atom by interacting with the O2 atom of reducing end sugars for formation of the cis-enediol intermediate. Although the roles of the three most important histidine residues have been proposed, several key aspects of its catalytic mechanism remain unclear. First, structural insights into deprotonation and subsequent protonation of the chiral center the C2 atom, which proceed in the open form of the sugar, are unclear because there is a lack of structural information regarding enzymes belonging to the AGE superfamily in complex with the open form of the sugar. Second, it is unclear how CE forms cis-enediol or cis-enediolate intermediates via the ring opening step, followed by ring closure. Important residues responsible for ring opening/closure of the substrate were confirmed in SeYihS and phosphoglucose isomerase (PGI) from Lactococcus lactis (LlPGI) (
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ,
      • Solomons J.T.
      • Zimmerly E.M.
      • Burns S.
      • Krishnamurthy N.
      • Swan M.K.
      • Krings S.
      • Muirhead H.
      • Chirgwin J.
      • Davies C.
      The crystal structure of mouse phosphoglucose isomerases at 1.6 Å resolution and its complex with glucose 6-phosphate reveals the catalytic mechanism of sugar ring opening.
      ). Based on the structural analyses of LlPGI and SeYihS, the nearby base catalyst (LlPGI-Lys-518 and SeYihS-Glu-251) was suggested to abstract a proton from the 1-OH of the substrate, with the histidine residue (LlPGI-His-388 and SeYihS-His-383) donating a proton to the O5 atom of the substrate as an acid catalyst. SeYihS-His-383 was, therefore, regarded as a bifunctional residue responsible for deprotonation of the O1 atom to form the cis-enediol intermediate as well as protonation of the O5 atom of d-mannose during ring opening (
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ). However, whether RaCE-His-374 is involved in ring opening as well as cis-enediol intermediate formation is still controversial. Finally, the importance of a flexible loop close to the catalytic site of CE is controversial due to the lack of structures in complex with its substrates/products. In the case of SeYihS, a flexible loop (β11-α8 loop) was shown to participate in binding to the substrate, d-mannose. Indeed, the conformation of this loop was changed upon substrate binding, and Arg-238 and Phe-239 residing in this loop was brought inward to the catalytic site to interact with d-mannose (
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ). In the case of RaCE, a flexible loop (β7-β8 loop) close to the catalytic site may compensate for the role of the β11-α8 loop in SeYihS (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ). Thus, comparing the presence or absence of its substrate is required to solve the problems noted here.
      In this study, we determined the three-dimensional structures of RmCE in complex with its substrates/products, 4-O-β-d-glucosyl-d-mannose (RmCE-Glc-Man) and epilactose (RmCE-epilactose) as closed form ligands, and with the intermediate analog, cellobiitol (RmCE-cellobiitol) as an open form ligand, as well as its apo-structure. These structures reveal the manner of interaction with ligands, and provide static snapshots of different states along the reaction pathway. Consequently, we propose the catalytic mechanism of this enzyme. In addition, we discuss important structural elements for high thermal stability of RmCE with regard to the structural features and sequence comparison with other CEs.

      EXPERIMENTAL PROCEDURES

      Preparation of Recombinant RmCE

      Recombinant RmCE was produced in Escherichia coli, and E. coli proteins were removed by heat treatment as described previously (
      • Ojima T.
      • Saburi W.
      • Sato H.
      • Yamamoto T.
      • Mori H.
      • Matsui H.
      Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.
      ). The samples thus obtained were further purified by anion exchange column chromatography using DEAE-Sepharose CL-6B (Amersham Biosciences) equilibrated with 50 mm Tris-HCl buffer (pH 8.5) to remove nucleic acid compounds. The flow-through fractions containing the samples were collected and dialyzed against 50 mm Tris-HCl buffer (pH 8.5) and 150 mm NaCl. Homogeneity of the samples was assessed by SDS-PAGE.

      Crystallization and Data Collection

      The purified RmCE solubilized in 50 mm Tris-HCl buffer (pH 8.5) and 150 mm NaCl was concentrated at 7.5 mg ml−1 using Vivaspin-4 10K (GE Healthcare), and subsequently used for initial crystallization screening using a series of crystallization kits from Qiagen (Hilden, Germany) by the sitting-drop vapor-diffusion method. Briefly, 0.5 μl of protein solution was mixed with an equal volume of reservoir solution. Initial crystals of RmCE were obtained with reservoir solution containing 0.1 m sodium acetate buffer (pH 4.5) and 1.0 m ammonium hydrogen phosphate. The initial conditions were then optimized to 0.1 m sodium acetate buffer (pH 4.5) and 1.2 m ammonium hydrogen phosphate using an aliquot of protein solution concentrated at 5.0 mg ml−1 in 30 mm Tris-HCl buffer (pH 8.5) and 60 mm NaCl. The crystals were grown to approximate dimensions of 0.1 × 0.1 × 0.05 mm within 5 days at 20 °C. For data collection, the crystals were soaked in cryoprotectant solution containing 20% (v/v) glycerol by repeated addition of 1.0 μl of cryoprotectant solution and discarding the 1.0-μl drop solution three times, followed by flash-cooling under a stream of liquid nitrogen at 100 K. Diffraction data were collected on beamline BL-41XU at SPring-8 (Hyogo, Japan). The asymmetric unit contained one molecule corresponding to a Matthews coefficient (
      • Kantardjieff K.A.
      • Rupp B.
      Matthewes coefficient probabilities. improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals.
      ) of 1.83 Å3 Da−1 and an estimated solvent content of 32.7%. To prepare the crystals of RmCE complexed with ligands, a single crystal was soaked in cryoprotectant solution containing 40% (w/v) sucrose and 10 mm cellobiose, 10 mm lactose, or 10 mm cellobiitol for 9 h at 20 °C. Diffraction data of each complex were collected on beamlines BL-1A and NW-12A at Photon Factory (Tsukuba, Japan). Data sets for RmCE in complex with each ligand were processed using the XDS program suite (
      • Kabsch W.
      XDS.
      ), whereas data sets for RmCE were processed using the HKL2000 program suite (
      • Otwinowski Z.
      • Minor W.
      Processing of X-ray diffraction data collected in oscillation mode.
      ). All data collection statistics are summarized in Table 1.
      TABLE 1Data collection and refinement statistics
      RmCERmCE-Glc-ManRmCE-epilactoseRmCE-cellobiitol
      Data collection
      Wavelength (Å)1.00001.00001.00001.0000
      Space groupP212121P212121P212121P212121
      Unit cell parameters (Å)a = 41.9a = 41.9a = 41.9a = 41.6
      b = 87.5b = 87.7b = 87.5b = 87.0
      c = 94.0c = 94.2c = 94.0c = 93.3
      Resolution (Å)50.00–1.74 (1.81–1.74)50.00–1.47 (1.56–1.47)50.00–1.64 (1.74–1.64)50.00–2.19 (2.31–2.19)
      Rmerge
      Rmerge = ΣhklΣi|Ii(hkl)−〈Ii(hkl)〉|/ΣhklΣiIi(hkl), where i is the number of observations of a given reflection and I(hkl) is the average intensity of the i observations. Rfree was calculated with a 5% fraction of randomly selected reflections evaluated from refinement. The highest resolution shell is shown in parentheses.
      0.101 (0.448)0.076 (0.511)0.071 (0.486)0.112 (0.586)
      Redundancy10.6 (9.1)6.4 (6.5)6.5 (6.4)7.1 (7.0)
      Completeness (%)99.9 (99.8)99.8 (99.2)99.0 (93.6)99.4 (96.6)
      Number of unique reflections36127 (3531)59748 (9460)42413 (6411)18054 (2775)
      I/σ(I)〉22.8 (4.0)16.8 (3.8)17.3 (3.6)14.6 (3.8)
      Refinement
      Resolution (Å)32.0–1.7439.8–1.4743.8–1.6438.1–2.19
      Rwork/Rfree0.158/0.1850.167/0.1940.160/0.1940.161/0.224
      Atoms
      Protein3317334533453317
      Water358611498231
      PO410555
      Cl ion1111
      Glc (ring form)11
      Glc (linear form)12
      Glc-Man23
      Epilactose23
      RMSD
      Bond length (Å)0.0060.0060.0060.007
      Bond angle (°)1.0881.1151.1021.078
      Ramachandran (%)
      Favored98.898.898.898.8
      Allowed1.21.21.21.2
      Outliers0000
      Mean B factor (Å2)
      Protein13.9412.1318.7323.37
      Solvent26.5524.5928.6430.06
      a Rmerge = ΣhklΣi|Ii(hkl)−〈Ii(hkl)〉|/ΣhklΣiIi(hkl), where i is the number of observations of a given reflection and I(hkl) is the average intensity of the i observations. Rfree was calculated with a 5% fraction of randomly selected reflections evaluated from refinement. The highest resolution shell is shown in parentheses.

      Structure Determination and Refinement

      The structures of RmCE and ligand-bound RmCE were determined by the molecular replacement method with the program AutoMR in the Phenix program package (
      • Adams P.D.
      • Afonine P.V.
      • Bunkoczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). The structure of apo-RmCE was solved using the structure of RaCE (PDB code 3VW5) as a search model. Subsequently, ligand-bound RmCE was determined using the structure of apo-RmCE as a search model. Rotation and translation functions were calculated using data of 45.0 to 3.0-Å resolution. Several rounds of refinement were performed using the program Phenix.refine in the Phenix program suite, alternating with manual fitting and rebuilding based on 2FoFc and FoFc electron densities using COOT (
      • Emsley P.
      • Cowtan K.
      COOT. Model-building tools for molecular graphic.
      ). Then, water molecules, metal ions, monophosphates, substrates/products (Glc-Man and epilactose), and intermediate analog (cellobiitol) were built based on 2FoFc and FoFc electron densities. The final refinement statistics and geometry defined by MolProbity (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity. All-atom structure validation for macromolecular crystallography.
      ) are summarized in Table 1.

      RESULTS AND DISCUSSION

      Overall Structures of RmCE in the Apo Form and in Complex with Ligands

      The structure of the apo-RmCE was determined by the molecular replacement method using the structure of RaCE as a search model and refined to 1.74-Å resolution. As shown in the other epimerases/isomerases belonging to the AGE superfamily (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ,
      • Lee Y.C.
      • Wu H.M.
      • Chang Y.N.
      • Wang W.C.
      • Hsu W.H.
      The central cavity from the (α/α)6 barrel structure of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase contains two key residues for reversible conservation.
      ,
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ,
      • Itoh T.
      • Mikami B.
      • Maru I.
      • Ohta Y.
      • Hashimoto W.
      • Murata K.
      Crystal structure of N-acetyl-d-glucosamine 2-epiemrase from porcine kidney at 2.0 Å resolution.
      ), the overall fold of RmCE was (α/α)6 barrel (Fig. 1A). The final refined model was comprised of RmCE monomer (residues Thr-3–Val-409), 358 water molecules, one chloride ion, and two phosphate ions. The chloride and phosphate ions would be derived from the buffers used for purification and crystallization of the recombinant RmCE. The chloride ion was located in the vicinity of the catalytic site, and coordinated by the amino group of the peptide backbone of residues Gly-387, Tyr-389, and His-390 (Fig. 1B). One of the phosphate ions occupied the substrate binding site surrounded by the inner six helices of the (α/α)6 barrel-fold, the other was bound to the positively charged patch formed by Arg-352, Arg-89, and Arg-96 in the symmetry-related molecule (Fig. 1, C and D). The latter phosphate ion, therefore, may affect crystal packing. Although cellobiose and lactose were used as substrates to obtain the complex structures by the crystal soaking method, electron densities of the epimerized β-anomeric Glc-Man and epilactose were clearly observed in the catalytic site (Fig. 2, A–D), suggesting that RmCE turned over these disaccharides in the crystal. Although the activity of RmCE is very low under the conditions of the crystal soaking experiment (pH 4.5, 20 °C), a soaking time of 9 h would have been sufficient for epimerization to proceed. Indeed, it is well known that catalytic reactions of several proteins can be performed in the crystal (
      • Mozzarelli A.
      • Rossi G.L.
      Protein function in the crystal.
      ,
      • Yamane J.
      • Ohyabu N.
      • Yao M.
      • Takemoto H.
      • Tanaka I.
      In-crystal chemical ligation for lead compound generation.
      ). In addition, selective binding to cellobiose or lactose was superior to that to sucrose (2-O-β-d-fructofuranosyl-α-d-glucoside) in the cryoprotectant as observed despite >100-fold greater concentration (40% (w/v) = 1.17 m) compared with cellobiose or lactose. The structures of RmCE-Glc-Man, RmCE-epilactose, and RmCE-cellobiitol were determined and refined to 1.47-, 1.64-, and 2.19-Å resolutions, respectively. The residues Thr-3–Arg-412 of RmCE-Glc-Man and RmCE-epilactose were built, while residues Thr-3–Val-409 of RmCE-cellobiitol were built. The chloride ion and the phosphate ions found in the molecular surface but not in the catalytic site were coordinated in the same fashion as observed in apo-RmCE. The overall structures of RmCE-Glc-Man, RmCE-epilactose, and RmCE-cellobiitol were very similar to that of apo-RmCE with root mean square deviation values of 0.139 Å for 374 Cα atoms, 0.125 Å for 374 Cα atoms, and 0.149 Å for 380 Cα atoms, respectively. Thus, no noticeable rearrangement was induced upon ligand binding.
      Figure thumbnail gr1
      FIGURE 1A, overall structure of apo-RmCE displayed as a ribbon diagram. B, chloride ion binding site. The residues around the bound chloride ion are represented as sticks. Chloride ion and water molecule are represented as spheres in red and green, respectively. Polar interactions are shown by dotted lines. Electron density of the bound chloride ion and water in the omit |Fo| − |Fc| map (gray) are calculated without the ligand and contoured at 3.0 σ. C and D, phosphate ion binding site. The residues around the bound phosphate ions are represented as sticks. Polar interactions are shown by dotted lines. Electron density of the bound phosphate in the omit |Fo| − |Fc| map (gray) are calculated without the ligand and contoured at 3.0 σ.
      Figure thumbnail gr2
      FIGURE 2A, C, and D, the overall structures of RmCE-Glc-Man, RmCE-epilactose, and RmCE-cellobiitol, respectively, displayed as ribbon diagrams. B, D, and F, stereo views of catalytic site architecture of RmCE-Glc-Man, RmCE-epilactose, and RmCE-cellobiitol, respectively. The residues involved in substrate recognition are represented as lines. Water molecules are represented as spheres (red). Electron densities of the bound Glc-Man, epilactose, and cellobiitol in the omit |Fo| − |Fc]bar] map (gray) were calculated without the substrate and contoured at 3.0 σ.

      Binding Manner of Closed Form Ligands

      In the structures of RmCE in complex with the closed form ligands, several residues contribute to ligand recognition. The catalytic site was formed by residues in the β5-β6 loop, Ser-185, and Asp-188, as well as those in the inner six helices, Arg-66, Tyr-124, Asn-196, His-200, His-259, Glu-262, Tyr-307, Trp-322, Trp-385, and His-390 (Fig. 2, B and D). Aromatic residues are generally responsible for sugar recognition by stacking interaction (
      • Asensio J.L.
      • Ardá A.
      • Cañada F.J.
      • Jiménez-Barbero J.
      Carbohydrate-aromatic interactions.
      ). The indole groups of Trp-322 and Trp-385 were stacked onto pyranose rings at the reducing and non-reducing ends of the ligands, respectively. Trp-322 of RmCE is completely conserved in enzymes belonging to the AGE superfamily, including AGEs and YihSs, whereas Trp-385 of RmCE is only conserved in CEs. This observation is consistent with the results of mutational analyses indicating that these two tryptophan residues are important for ligand binding (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ,
      • Ito S.
      • Hamada S.
      • Ito H.
      • Matsui H.
      • Ozawa T.
      • Taguchi H.
      • Ito S.
      Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus.
      ). Moreover, the side chains of amino acid residues and water-mediated hydrogen bonds also recognize hydroxyl groups of ligands (Fig. 3A). Trp-322 recognizes the O2 atom of the d-mannose residue at the non-reducing end through an ordered water molecule. The amino groups of His-320 and Trp-322 help to position the water molecule regardless of ligand binding, suggesting that water is always trapped in the enzymes to orient the disaccharide properly in the catalytic site. Arg-66, which is completely conserved in the AGE superfamily and is essential for the catalytic activity (
      • Asensio J.L.
      • Ardá A.
      • Cañada F.J.
      • Jiménez-Barbero J.
      Carbohydrate-aromatic interactions.
      ), makes direct contact with the O6 atom of the d-mannose residue. Such an interaction was also observed in d-mannose-bound SeYihS (
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ). Extensive interactions were also identified around the reducing end sugar through Trp-265, Glu-326, and Arg-393, which are the other catalytically important residues confirmed by mutational analyses (
      • Asensio J.L.
      • Ardá A.
      • Cañada F.J.
      • Jiménez-Barbero J.
      Carbohydrate-aromatic interactions.
      ), and formed a salt bridge and hydrogen bond network to optimize the orientation and charge of His-390 and Glu-262 (Fig. 3A).
      Figure thumbnail gr3
      FIGURE 3A, contact between Glc-Man or epilactose and RmCE. The distances between specific atoms are shown in Å as dotted lines. Numbers in parentheses are for contact between epilactose and RmCE. B, contact between cellobiitol and RmCE. The distances between specific atoms are shown in Å as dotted lines.
      In addition to common interactions between two structures in complex with closed form ligands, the non-reducing end sugar mediates possible hydrogen bonding with Ser-185 and Asp-188 (Fig. 4A) in the β5-β6 loop positioned close to the active site. Ser-185 is completely conserved in known CEs characterized to date, whereas Asp-188 is diverse (Fig. 4B). In the RmCE-Glc-Man structure, the O4 atom of the glucosyl residue at the non-reducing end directly forms hydrogen bonds with the side chain of Ser-185. In contrast, Ser-185 is slightly far from its O4 atom in the RmCE-epilactose structure, which roughly correlates with high affinity toward cellobiose rather than lactose in most CEs except for Dyadobacter fermentans CE (DfCE), Flavobacterium johnsoniae CE (FjCE), and Pedobacter heparinus CE (PhCE) (
      • Ojima T.
      • Saburi W.
      • Yamamoto T.
      • Mori H.
      • Matsui H.
      Identification and characterization of cellobiose 2-epimerases from various aerobes.
      ). On the other hand, diverse Asp-188 was positioned within hydrogen bonding distance to the O6 atom of both glucosyl and galactosyl residues of ligands. However, there is no relationship between the amino acid corresponding to Asp-188 and affinity toward cellobiose and lactose among CEs. One of the most striking aspects is that conformation of the β5-β6 loop in RmCE is invariant independent of ligand binding, whereas the corresponding β11-α8 loop in SeYhiS with a high degree of flexibility is visible in the catalytic site upon ligand binding (Fig. 4C).
      Figure thumbnail gr4
      FIGURE 4A, the structure of apo-form of RmCE is superposed on that of Glc-Man-bound RmCE (upper panel), and that of epilactose (lower panel). Apo structures in both panels are shown in light green. The β5-β6 loops in RmCE in complex with Glc-Man (blue) and epilactose (dark green) are represented and the residues close to the substrate are depicted as sticks. B, multiple sequence alignment of several CEs. Serine and aspartic acid residues related to substrate binding are indicated by stars and solid circles, respectively. The multiple sequence alignment was performed using the programs ClustalW and ESPript. The sequences are as follows (GenBankTM accession numbers in parentheses): RmCE (BAK61777.1), RaCE (BAF81108.1), BfCE (BAH23773.1), EcCE (BAG68451.1), CsCE (WP_011915904.1), and DtCE (BAM66298.1). C, the ribbon diagrams of β11-α8 loop in SeYihS with or without d-mannose are shown in green and blue, respectively. The residues of the orientations of which are changed to make contact with the ligand are depicted by sticks.

      Binding Manner of Open Form Ligand

      The structure in complex with the reaction intermediate analog was required to understand the reaction mechanism of CE. Cellobiitol is an open chain form, and it could be an analog of the reaction intermediate. As shown in Fig. 2, E and F, this enzyme-intermediate analog complex structure is the first structural evidence for the active site among the enzymes belonging to the AGE superfamily. In the structure of RmCE-cellobiitol, the pyranose ring of cellobiitol was recognized in an orientation similar to glucosyl and galactosyl residues at the non-reducing end of bound Glc-Man and epilactose, respectively (Figs. 2F and 3B). Moreover, the relative orientation of the d-glucitol part of cellobiitol was nearly identical to the reducing end sugar of Glc-Man and epilactose except for the positions of its C1 and O1 atoms (Fig. 5A). Projection of this d-glucitol part seen along with the C2-C3 bond indicated an eclipsed-like conformation with pseudoaxial orientation of the O2 atom (Fig. 5, B and C). The O2 atom of the d-glucitol part was turned around the C2-C3 bond about 90° from the equatorial position of the O2 atom of the ideal cellobiose. As a result, the C1-C2 bond of the d-glucitol part of cellobiitol was positioned between two His residues (His-259 and His-390) (Fig. 5A). These histidine residues correspond to RaCE-His-243 and RaCE-His-374, considered as acid/base catalysts during the epimerization step via the cis-enediol intermediate. On the basis of these observations, we propose that bound cellobiitol mimics the state just before abstraction of the H2 proton.
      Figure thumbnail gr5
      FIGURE 5A, relative orientations of three catalytic histidine residues to cellobiitol and Glc-Man. Residues in RmCE-cellobiitol and RmCE-Glc-Man are represented as sticks in yellow and blue, respectively. B, the closed form sugars at the reducing end of cellobiose (left) and Glc-Man (right), and the sugar alcohol part of cellobiitol (middle) viewed along with the C2-C3 bond. C, projection of the closed form sugars at the reducing end of cellobiose (left) and Glc-Man (right), and the sugar alcohol part of cellobiitol (middle) viewed along with the C2-C3 bond.

      Structural Insights into the Ring Opening/Closure Step of the Catalytic Reaction with RmCE

      The architectures of catalytic sites as well as the overall fold of RmCE are similar to those of AGEs and YihSs, suggesting that these enzymes share a similar catalytic mechanism. The catalytic mechanism for CE was recently proposed based on sequence and structure comparison with AGEs and YihSs, as well as biochemical data (
      • Fujiwara T.
      • Saburi W.
      • Inoue S.
      • Mori H.
      • Matsui H.
      • Tanaka I.
      • Yao M.
      Crystal structure of Ruminococcus albus cellobiose 2-epimerase. Structural insights into epimerization of unmodified sugar.
      ,
      • Asensio J.L.
      • Ardá A.
      • Cañada F.J.
      • Jiménez-Barbero J.
      Carbohydrate-aromatic interactions.
      ). Understanding the details of the reaction mechanism of CE, however, was restricted to the deprotonation/reprotonation process to form cis-enediol intermediates. Successfully determined crystal structures of RmCE in complex with a series of ligands yielded further insights into interconversion of the d-glucose and d-mannose residues of disaccharide with RmCE. Here, we provide the reaction mechanism proceeding via ring opening/closure and deprotonation/reprotonation coupled with rotation of the carbon-carbon bond to form the cis-enediol intermediate (Fig. 6).
      Figure thumbnail gr6
      FIGURE 6Schematic diagram of the proposed epimerization catalyzed by RmCE. The electron transfer processes to convert glucose into mannose and its reverse conversion are represented by black and gray arrows, respectively.
      Binding of cellobiitol to RmCE reflects the requirement of ring opening to undergo epimerization. In addition, ring opening is a common process for the first step of sugar isomerization/epimerization, as exemplified by phosphoglucose isomerase, galactose mutarotase, and rhamnose isomerase (
      • Solomons J.T.
      • Zimmerly E.M.
      • Burns S.
      • Krishnamurthy N.
      • Swan M.K.
      • Krings S.
      • Muirhead H.
      • Chirgwin J.
      • Davies C.
      The crystal structure of mouse phosphoglucose isomerases at 1.6 Å resolution and its complex with glucose 6-phosphate reveals the catalytic mechanism of sugar ring opening.
      ,
      • Thoden J.B.
      • Kim J.
      • Raushel F.M.
      • Holden H.M.
      The catalytic mechanism of galactose mutarotase.
      ,
      • Yoshida H.
      • Yoshihara A.
      • Teraoka M.
      • Yamashita S.
      • Izumori K.
      • Kamitori S.
      Structure of l-rhamnose isomerase in complex with l-rhamnopyranose demonstrates the sugar-ring opening mechanism and the role of a substrate sub-binding site.
      ) as well as aAGE and SeYihS (
      • Lee Y.C.
      • Wu H.M.
      • Chang Y.N.
      • Wang W.C.
      • Hsu W.H.
      The central cavity from the (α/α)6 barrel structure of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase contains two key residues for reversible conservation.
      ,
      • Itoh T.
      • Mikami B.
      • Hashimoto W.
      • Murata K.
      Crystal structure of YihS in complex with d-mannose. Structural annotation of Escherichia coli Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerases.
      ). The proposed mechanisms indicated that acid (A–H) and base (B:) catalysts are involved in ring opening/closure by donating a proton to the O5 atom of the reducing end moiety of the substrate and abstracting the proton from the O1 atom of the same moiety, respectively. As a consequence, the C1-O5 bond of the reducing end sugar was cleaved. The only candidate for the acid catalyst is His-390, as the basic residue Arg-66 also located within hydrogen bond distance to the O5 atom (3.3 Å) (Fig. 3A) is unlikely to act as an acid catalyst due to its high theoretical pKa value, whereas those for the base catalysts are His-390, Glu-262, and His-200.
      The Nϵ2 atom of His-390 was close to both O5 and O1 atoms of the reducing end of substrates, within 2.9 and 3.0 Å, respectively (Fig. 3A), which are reasonable for His-390 to serve as an acid/base catalyst for ring opening. In the direction of Glc → Man, initially deprotonated His-390 abstracts the proton from the O1 atom of the reducing end d-glucose residue, and then the resulting O5 anion accepts a proton from the protonated His-390, in agreement with the following role of His-390 as the base catalyst to form the cis-enediol intermediate (Fig. 7A). In the opposite direction of epimerization, initially protonated His-390 donates a proton to the O5 atom, and simultaneously His-390 abstracts the proton from the O1 atom, in agreement with the following role of His-390 as the acid catalyst to donate a proton to the cis-enediol intermediate (Fig. 7A).
      Figure thumbnail gr7
      FIGURE 7A, schematic diagram of the proposed ring opening mechanism involving His-390. The electron transfer reaction is represented by arrows (black). B, schematic diagram of the proposed proton-relay system. The electron transfer processes of forward and reverse reactions are represented by black and gray arrows, respectively.
      The mechanism of ring closure can be interpreted as the inverse reaction of ring opening. After the deprotonation/reprotonation step, rotation about the C2-C3 bond may occur again to bring the O1 atom of the reducing end moiety of the substrate close to the O5 atom of the same moiety (Fig. 6). Then, the electron pair on the O5 anion caused by deprotonation with His-390 attacks the electron-deficient C1 atom to remake the C1-O5 bond. Thus, this ring opening/closure mechanism involving only His-390 has no inconsistency for epimerization reactions in both directions.
      On the other hand, it remains possible that Glu-262 or His-200 are related to ring opening/closure as a general base catalyst. However, the Oϵ1 atom of Glu-262 was slightly apart from the O1 atom within 3.5 Å (Fig. 3A), although it was proposed that the corresponding His-Glu pair in SeYihS (His-383–Glu-251) was involved in ring opening. His-200 is within 3.2 Å from the C1 atom of the reducing end of substrates (Fig. 3A), but this residue is not conserved in AGEs.
      His-390 may be deprotonated when Glu-262 or His-200 acts as the base catalyst in ring opening. In the direction of Man → Glc, therefore, deprotonated His-390 should be protonated to donate a proton to the cis-enediol intermediate as the acid catalyst. When the open form of ligands is closed by the reverse reaction of ring opening, His-390 should be deprotonated to abstract the proton from the O5 atom of the reducing end part of the substrate as the base catalyst. In the direction of Glc → Man, therefore, His-390 protonated by abstracting a proton to form the cis-enediol intermediate should be deprotonated to act as the base catalyst for sequential ring closure. If His-390 acts as the acid catalyst for ring opening, therefore, there must be a system to remove the inconsistency on the charged state of His-390. A possible mechanism for exchanging the charged states of His-390 is that a proton may be relayed via nearby Glu-326, and Arg-393, forming a polar interaction network together with His-390 (Fig. 7B).

      Structural Insights into the Deprotonation/Reprotonation Step of the Catalytic Reaction with RmCE

      As mentioned previously (
      • Amein M.
      • Leatherwood J.M.
      Mechanism of cellobiose 2-epimerase.
      ), the H2 proton of the reducing end part of the substrate should be abstracted to form a cis-enediol intermediate after ring opening. The binding structure of the open form of the intermediate analog cellobiitol allowed us to deduce the detailed mechanism of epimerization by CE. Considering the stereochemistry of cellobiitol (Fig. 5A), the H2 proton of the d-glucitol part is close to His-390 by rotation about the C2-C3 bond, whereas it is very difficult for His-390 to approach the same H2 proton in the structure of RmCE in complex with closed form ligands. When converting the d-glucose residue at the reducing end of substrates to the d-mannose residue, His-390 is the most feasible candidate to abstract the H2 proton from the reducing end d-glucose residue after rotation about the C2-C3 bond. When the proton is supplied from the opposite side of the general base catalyst His-390 across the C1=C2 double bond in the state of cis-enediol intermediate, the configuration around the stereogenic center of C2 converts. The orientation of the C1=C2 double bond that results in rotation about the C2-C3 bond makes it possible that the C2 atom accepts a proton from the protonated His-259 as a general acid catalyst. In this situation, His-200, corresponding to RaCE-His-184, maintained in close proximity to the 2-OH group of the d-glucitol part by about 2.9 Å, fulfills the proposed roles of RaCE-His-184 as assistant to depolarization of the 2-OH group. The distance between the Nϵ2 atom of His-200 and the O1 atom of the d-glucitol part of cellobiitol was also about 3.0 Å, suggesting that the negatively charged enolate anion of the actual intermediate is stabilized by the positive charge on His-200 (Fig. 5A).
      On the other hand, the H2 proton of the d-mannose residue at the reducing end was close to His-259 in the structure of RmCE in complex with the closed form ligands. When converting this d-mannose residue to d-glucose residue, therefore, His-259 is likely to abstract the H2 proton from the d-mannose residue independent of rotation about the C2-C3 bond. Subsequently, to convert the configuration of the C2 atom, the C2 atom accepts a proton from the opposite side of His-259 across to the C1=C2 double bond, where His-390 is located. Hence, His-390 is a reasonable candidate to function as an acid catalyst in this direction, and agreement with their roles proposed as reversible general acid/base catalysts in epimerization. Taken together, our results strongly suggest the requirement of rotation about the C2-C3 bond coupled with deprotonation/reprotonation to achieve reversible epimerization associated with three His residues (His-200, His-259, and His-390).
      Taken together, the structural studies of RmCE presented here provide molecular insights into the reaction mechanism for CE partly postulated based on structure analysis of the apo-form of RaCE. Further studies, such as neutron diffraction analyses exemplified by xylose isomerase (
      • Kovalevsky A.Y.
      • Hanson L.
      • Fisher S.Z.
      • Mustyakimov M.
      • Mason S.A.
      • Forsyth V.T.
      • Blakeley M.P.
      • Keen D.A.
      • Wagner T.
      • Carrell H.L.
      • Katz A.K.
      • Glusker J.P.
      • Langan P.
      Metal ion roles and the movement of hydrogen during reaction catalyzed by d-xylose isomerases. A joint X-ray and neutron diffraction study.
      ) and ultra-high resolution crystal structure analysis, would clarify the proton transfer and charged states of catalytic residues in catalysis by visualizing hydrogen atoms.

      Relationship between Thermostability and Structure in RmCE

      The overall fold of RmCE was similar to that of RaCE with a root mean square deviation of 0.88 Å for 338 Cα atoms. Although the conformations of both enzymes are almost identical, their α1 and α12 helices have different lengths; the α1 and α12 helices of RmCE are longer than those of RaCE by 1.5 turns each (Fig. 8A). Especially, five Leu residues in this extended 1.5 turns of helices α1 and α12 (Leu-12, Leu-15, Leu-402, Leu-405, and Leu-406) participated in forming a hydrophobic core with other hydrophobic residues, Val-19, Leu-328, Leu-332, Tyr-335, Tyr-344, Ala-347, Trp-351, and Ala-398 (Fig. 8A). The hydrophobic interactions observed in the buried surface area generally contribute to entropic stabilization of proteins (
      • Kellis Jr., J.T.
      • Nyberg K.
      • Sali D.
      • Fersht A.R.
      Contribution of hydrophobic interactions to protein stability.
      ). In addition, the contents of Pro and Arg residues in RmCE are much higher than those in other CEs (Table 2), although Caldicellulosiruptor saccharolyticus CE (CsCE) and Dictyoglomus turgidum CE (DtCE) have less Pro and Arg residues regardless of their high optimum temperatures. Twenty Pro residues (1.7–5.0 times more than the number of these residues in other CEs described above) were situated in the loops or the terminals of helices of RmCE, and contribute to restricting the flexibility of the peptide backbone (Fig. 8B). It is assumed that reduced flexibility of unstructured regions is one of the reasons for the acquisition of thermostability by this enzyme (
      • Takano K.
      • Higashi R.
      • Okada J.
      • Mukaiyama A.
      • Tadokoro T.
      • Koga Y.
      • Kanaya S.
      Proline effect on thermostability and slow unfolding of a hyperthermophilic protein.
      ). The content of the other key residue, Arg, in RmCE is also 1.8–6.6 times higher than in the other CEs. The higher frequency of Arg residues in the exposed state is one of the critical factors related to protein thermal stability (Fig. 8B) (
      • Pack S.P.
      • Yoo Y.J.
      Protein thermostability. Structure-based difference of amino acid between thermophilic and mesophilic proteins.
      ). This feature has been explained by the observation that Arg residues participate in formation of pairs with negatively charged amino acid residues, such as Asp and Glu residues, on the molecular surface. As a result, hydrophobic interactions in the buried surface area and hydrophilic interactions on the molecular surface may contribute to the thermal stability of RmCE.
      Figure thumbnail gr8
      FIGURE 8Relationships between structural features to thermal stability of RmCE. The structure displayed is the Glc-Man-bound form of RmCE. A, hydrophobic core formed by helices α1, α10, α11, and α12. Ribbon diagrams of RmCE (upper panel) and RaCE (lower panel) are shown, and hydrophobic residues are represented as sticks. Extended parts of helices in RmCE are shown in dark blue. B, positions of Pro and Arg residues in RmCE (upper left and upper right, respectively) and RaCE (lower left and lower right, respectively) are indicated. Surface representations of Pro and Arg residues are shown in orange and blue, respectively.
      TABLE 2Relationships between the compositions of prolines and arginines and optimum temperature in several CEs
      ProlineArginineOptimum temperature (° C)
      RmCE20 (4.9%)46 (11.2%)80
      CsCE4 (1.0%)15 (3.8%)75
      DtCE11 (2.8%)12 (3.1%)70
      DfCE12 (3.1%)16 (4.1%)50
      BfCE8 (2.0%)25 (6.4%)45
      SlCE18 (4.5%)18 (4.5%)45
      HaCE14 (3.4%)21 (5.0%)45
      TtCE10 (2.4%)23 (5.4%)35
      EcCE9 (2.2%)18 (4.4%)35
      SdCE8 (2.0%)16 (3.9%)35
      PhCE7 (1.8%)14 (3.5%)35
      FjCE8 (2.0%)7 (1.8%)35
      RaCE6 (1.5%)16 (4.1%)30

      Acknowledgments

      We thank the staff of beamline BL-41XU at SPring-8, and BL-1A and NW-12A at the Photon Factory for assistance with data collection.

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