Analysis of a New Family of Widely Distributed Metal-independent α-Mannosidases Provides Unique Insight into the Processing of N-Linked Glycans

The modification of N-glycans by alpha-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for alpha 1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-alpha 1,6-mannosidase activity consistent with specificity for N-linked glycans having their alpha 1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-Bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (alpha/alpha)(6)-fold, recognize and hydrolyze the alpha 1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-alpha 1,6-mannosidases.