Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

Abstract

A gene cluster involved in N -glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β- N -acetylhexosaminidase, exo -α-sialidase, and endo-β- N -acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl- N -acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N -glycan catabolism where β-1,4-d-mannosyl- N -acetyl-d-glucosamine is liberated from N -glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N -acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis , Bacteroides helcogenes , Bacteroides salanitronis , Bacteroides vulgatus , Prevotella denticola , Prevotella dentalis , Prevotella melaninogenica , Parabacteroides distasonis , and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N -glycans. A notable feature of the new metabolic pathway for N -glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl- N -acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N -glycans that includes a phosphorylase. We propose 4- O -β-d-mannopyranosyl- N -acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl- N -acetyl-d-glucosamine phosphorylase as the short name for BT1033. Background: N -Glycans are metabolized by sequential glycoside hydrolase-catalyzed reactions. Results: A phosphorylase encoded in a gene cluster involved in N -glycan metabolism in the genome of Bacteroides thetaiotaomicron catalyzed reversible phosphorolysis of β-1,4-d-mannosyl- N -acetyl-d-glucosamine. Conclusion: An N -glycan metabolic pathway containing a unique phosphorylase was discovered. Significance: B. thetaiotaomicron efficiently utilizes the energy of ATP via a phosphorylase-dependent metabolic pathway.