Synthesis of an allergy inducing tetrasaccharide "4P-X".

4P-X (ß-D-galactopyranosyl-(1 â†’ 4)-ß-D-galactopyranosyl-(1 â†’ 6)-[ß-D-galactopyranosyl-(1 â†’ 4)]-ß-D-glucopyranose) is included in galacto-oligosaccharides (GOSs) produced by ß-galactosidase derived from Bacillus circulans. 4P-X has been known to induce particularly strong allergies. High purity 4P-X is essential for use as a standard to quantify the amount of 4P-X in GOSs; however, the isolation of high purity 4P-X has never been reported. In this study, we achieved the synthesis of 4P-X by a combination of organic and enzymatic chemical syntheses in a short time. This is the first report of isolated, high purity 4P-X.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

In Ruminococcus albus, 4-O-ß-D-mannosyl-D-glucose phosphorylase (RaMP1) and ß-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze ß-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-ß-D-mannosyl-d-glucose and RaMP2 with/without ß-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.

Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-ß-D-mannosyl-D-glucose phosphorylase-like protein, UnkA.

The aerobic soil bacterium Cellvibrio vulgaris has a ß-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting D-mannose to D-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-ß-D-mannosyl-D-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and ß-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for ß-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for D-mannose. Recombinant UnkA phosphorolyzed ß-D-mannosyl-(1→4)-D-glucose and specifically utilized D-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-ß-D-mannosyl-D-glucose phosphorylase.

Characterization of a thermophilic 4-O-ß-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus.

4-O-ß-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-ß-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts ß-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-ß-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5-8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl ß-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.

Metabolic mechanism of mannan in a ruminal bacterium, Ruminococcus albus, involving two mannoside phosphorylases and cellobiose 2-epimerase: discovery of a new carbohydrate phosphorylase, ß-1,4-mannooligosaccharide phosphorylase.

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