Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (CE; EC 5.1.3.11), which converts cellobiose to 4-O-β-d-glucosyl-d-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE-producing bacterium, Bacteroides fragilis. In this pathway, β-1,4-mannobiose is epimerized to 4-O-β-d-mannosyl-d-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to α-d-mannosyl 1-phosphate (Man1P) and d-glucose by Man-Glc phosphorylase (MP; EC 2.4.1.281). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential Bi Bi mechanism. In a synthetic reaction, RaMP1 showed high activity only toward d-glucose and 6-deoxy-d-glucose in the presence of Man1P, whereas RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on d-glucose derivatives at the C2- and C3-positions, including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: β-linked glucobioses, maltose, N,N′-diacetylchitobiose, and β-1,4-mannooligosaccharides. Particularly, β-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than d-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward β-1,4-mannobiose but efficiently degraded β-1,4-mannooligosaccharides longer than β-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of β-1,4-mannooligosaccharides longer than β-1,4-mannobiose to produce Man1P and β-1,4-mannobiose.

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.

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2–10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.