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

Abstract

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-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 multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-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 α-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 α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.

Lactococcus lactis HkyuLL 10 suppresses colorectal tumourigenesis and restores gut microbiota through its generated alpha-mannosidase

“…23 Some gut commensal bacteria (eg, Bacteroides thetaiotaomicron, Enterococcus faecalis, Neobacillus novalis) also express αMAN to modulate or degrade yeast α-mannan and mannoserich glycans. [24][25][26] To ascertain the role of bacterial αMAN, we constructed a strain of E. coli that expressed the sequence of αMAN in HkyuLL 10. This mutant E. coli significantly inhibited the growth and proliferation of CRC cells and patient-derived organoids, thus confirming the tumour-suppressing function of HkyuLL 10-secreted αMAN.…”