Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

Abstract

Phosphorylases are a group of enzymes involved in formation and cleavage of glycoside linkage together with glycoside hydrolases and glycosyl-nucleotide glycosyltransferases (synthases). Phosphorylases, which reversibly phosphorolyze oligosaccharides to produce monosaccharide 1-phosphate, are generally intracellular enzymes showing strict substrate specificity. Physiologically, such strict substrate specificity is considered to be closely related to the environment containing bacteria possessing them. For example, D-galactosyl-␤133-N-acetyl-Dhexosamine phosphorylase (GalHexNAcP 2 ; EC 2.4.1.211) fromBifidobacterium longum, an intestinal bacterium, forms part of the pathway metabolizing galacto-N-biose (GNB; D-Gal-␤133-D-GalNAc) from mucin and lacto-N-biose I (LNB; D-Gal-␤133-D-GlcNAc) from human milk oligosaccharides, both of which are present in the intestinal environment, with GNB-and LNB-releasing enzymes and GNB/LNB transporter (1-8). Another example is cellobiose phosphorylase from Cellvibrio gilvus, which is a cellulolytic bacterium. Cellobiose phosphorylase forms an important cellulose metabolic pathway with an extracellular cellulase system producing cellobiose (9, 10). The reversible catalytic reaction of phosphorylases is one of the most remarkable features that make them suitable catalysts for practical syntheses of oligosaccharides. An oligosaccharide can be produced from inexpensive material by combining reactions of two phosphorylases, one for phosphorolyzing the material and the other for synthesizing the oligosaccharide, in one pot. Based on this idea, LNB is synthesized on a large (kg) scale using sucrose phosphorylase and GalHexNAcP (11). Practical synthesis methods of trehalose and cellobiose have also been developed (12, 13). However, only 14 kinds of substrate specificities have been reported among phosphorylases (13), thus restricting their use. Therefore, it would be useful to find a phosphorylase with novel activity.GalHexNAcP phosphorolyzes GNB and LNB to produce ␣-D-galactose 1-phosphate (Gal 1-P) and the corresponding N-acetyl-D-hexosamine. To date, GalHexNAcP is the only phosphorylase known to act on ␤-galactoside. This enzyme was first found in the cell-free extract of Bifidobacterium bifidum (14) and then in B. longum (1, 15), Clostridium perfringens (16), Propionibacterium acnes (17), and Vibrio vulnificus (18). These studies revealed that GalHexNAcPs were classified into three subgroups based on substrate preference between GNB and LNB. These subgroups are as follows: 1) galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP), showing similar activity on both GNB and LNB (B. longum and B. bifidum); 2) galacto-Nbiose phosphorylase (GNBP), preferring GNB to LNB (C. perfringens and P. acnes); and 3) lacto-N-biose I phosphorylase (LNBP), preferring LNB to GNB (V. vulnificus) (18). The ternary structure of GLNBP from B. longum (GLNBP Bl ) has been revealed recently (19). Based on the similarity in ternary structures between GLNBP Bl and ␤-galactosidase from Thermus

Diversity of phosphorylases in glycoside hydrolase families

“…1,4-β-Galactosyl-L-rhamnose phosphorylase (GalRhaP) was found in the genome of Clostridium phytofermentans alongside two GLNBPs (Nakajima et al 2009a). This enzyme produced Galβ1→ 3Glc and Galβ1→3Gal in the reverse reaction when using Glc and Gal, respectively, as acceptors with high K m values but did not use GlcNAc and GalNAc as acceptors.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…Since many genomic and metagenomic projects on human-related microbes are now in progress worldwide, 100) this information should be useful in predicting the possible substrate preferences of putative GalHexNAcP genes. Recently, Nakajima et al discovered a novel type of GH112 enzyme (D-galactosyl-1,4-L-rhamnose phosphorylase) in anaerobic bacteria, Clostridium phytofermentans 101) and Optitus terrae. 102) A new EC number (2.4.1.247) was given to it.…”

Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases

“…The GH112 family comprises phosphorlyases that act on β-D-galactosides to generate α-D-galactose 1-phosphate (αGal1P) (1), including 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (EC 2.4.1.211) 9, 10 and 1,4-β-D-galactosyl-L-rhamnose phosphorylase (EC 2.4.1.247) 11 . The GH130 family comprises phosphorylases that act on β-D-mannosides to generate α-D-mannose 1-phosphate (αMan1P) (2), including 1,4-β-mannosyl-glucose phosphorylase (EC 2.4.1.281) 12 , β-1,4-mannooligosaccharide phosphorylase (EC 2.4.1.319) 13 , and 1,4-β-mannosyl-N-acetylglucosamine phosphorylase (EC 2.4.1.320) 14,15 .…”