Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase

Abstract

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-beta-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, beta-1,4-mannobiose is epimerized to 4-O-beta-D-mannosyl-D-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to alpha-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: beta-linked glucobioses, maltose, N, N'-diacetylchitobiose, and beta-1,4-mannooligosaccharides. Particularly, beta-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than D-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward beta-1,4-mannobiose but efficiently degraded beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose to produce Man1P and beta-1,4-mannobiose.