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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.

      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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