Structural and functional characterization of a multi-domain GH92 alpha-1,2-mannosidase from Neobacillus novalis

摘要

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cellwall proteins, an extended alpha-1,6-mannan backbone carrying a number of alpha-1,2and alpha-1,3-mannose substituents of varying lengths. alpha-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the alpha-endomannanases, which then degrade the alpha-mannan backbone. Most characterized GH92 alpha-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multidomain GH92 alpha-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 alpha-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 alpha-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast alpha-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 alpha-mannosidases for the degradation of yeast alpha-mannan or mannose-rich glycans.