The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan

• Published in: The Journal of biological chemistry Vol. 290; no. 41; pp. 25023 - 25033
• Main Authors: Cuskin, Fiona, Baslé, Arnaud, Ladevèze, Simon, Day, Alison M., Gilbert, Harry J., Davies, Gideon J., Potocki-Véronèse, Gabrielle, Lowe, Elisabeth C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.10.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacteroides; Bacteroides - enzymology; Biocatalysis; Candida; Candida albicans; Catalytic Domain; Crystallography, X-Ray; enzyme; Enzymology; glycosidase; glycoside hydrolase; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - metabolism; Life Sciences; Mannans - chemistry; Mannans - metabolism; Mannose - chemistry; microbiome; Models, Molecular; Molecular Sequence Data; phosphorylase; Phosphorylases - chemistry; Phosphorylases - metabolism; Protein Binding; x-ray crystallography; enzyme; glycosidase; x-ray crystallography; glycoside hydrolase; microbiome; phosphorylase; X-ray crystallography; cell wall; gut symbiont; yeast
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.681460

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu227 and Glu268, hydrogen bond to O1 of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages. Background: A cohort of a family of mannose phosphorylases lack phosphate binding residues, suggesting that they display non-phosphorylase activities. Results: The non-phosphorylase enzymes were shown to be β-mannosidases. Conclusion: Replacing basic phosphate binding residues with carboxylic amino acids converts mannoside phosphorylases into glycoside hydrolases. Significance: Functional phylogeny can be used to distinguish between closely related glycan phosphorylases and glycoside hydrolases.