The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

Hidaka, Masafumi; Nishimoto, Mamoru; Kitaoka, Motomitsu; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya

doi:10.1074/jbc.m808525200

Cited by 49 publications

(34 citation statements)

References 64 publications

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“…2. Because catalytic and substrate recognition residues of GLNBP Bl have already been identified from its ternary structure (19), we compared the corresponding residues of these proteins. The catalytic proton donor of GLNBP Bl (Asp 313 ) was conserved in Cphy0577, Cphy1920, and Cphy3030 proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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…”

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confidence: 99%

“…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 thermophilus, which belongs to glycoside hydrolase family 42 (19,20), GalHexNAcP homologs are classified as GH112 (glycoside hydrolase family 112), although phosphorylases are glycosyltransferases (21,22).…”

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confidence: 99%

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Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

Nakajima

Nishimoto

Kitaoka

2009

Journal of Biological Chemistry

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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

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Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

Nakajima

Nishimoto

Kitaoka

2009

Journal of Biological Chemistry

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“…95) GLNBP is a homodimeric protein (Fig. 5A), and its structure consists of a catalytic domain and three additional domains: an Ig-like fold, an = fold, and a C-terminal -sheet domain.…”

Section: Glnbpmentioning

confidence: 99%

Unique Sugar Metabolic Pathways of Bifidobacteria

Fushinobu

2010

Bioscience, Biotechnology, and Biochemistry

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“…1). LNBP catalyzes the first reaction of this pathway, the phosphorolytic cleavage of a galactosyl-beta-1,3-N-acetylhexosamine (GNB or LNB) into an N-acetylhexosamine (HexNAc) and galactose 1-phosphate (gal1P) (14). Due to its similarity to the Leloir pathway and the direct generation of gal1P without action of a galactokinase (GalK), the GNB/LNB pathway is considered an energy-saving variant of the Leloir pathway (Fig.…”

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Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases

Bruyn

Beauprez

Maertens

et al. 2013

Appl Environ Microbiol

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The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

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The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

Cited by 49 publications

References 64 publications

Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

Unique Sugar Metabolic Pathways of Bifidobacteria

Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases

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