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

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 79; no. 5; pp. 387 - 400
• Main Authors: Kołaczkowski, Bartłomiej M., Moroz, Olga V., Blagova, Elena, Davies, Gideon J., Møller, Marie Sofie, Meyer, Anne S., Westh, Peter, Jensen, Kenneth, Wilson, Keith S., Krogh, Kristian B. R. M.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.05.2023; Wiley Subscription Services, Inc
• Subjects: alpha-Mannosidase - metabolism; Binding sites; Biodegradation; Carbohydrates; carbohydrate‐binding module family 32; Crystal structure; Domains; Glycan; glycans; glycoside hydrolase family 92; Mannan; Mannans - chemistry; Mannans - metabolism; Mannose; Mannose - chemistry; Mannose - metabolism; Mannosidase; Mannosidases - chemistry; Mannosidases - metabolism; Models, Molecular; Neobacillus; Neobacillus novalis; Oligosaccharides; Polysaccharides; Polysaccharides - chemistry; Proteins; Saccharomyces cerevisiae - metabolism; Structural analysis; Structural integrity; Structure-function relationships; Substrate Specificity; Substrates; Yeast; α‐mannosidases; glycoside hydrolase family 92; glycans; Neobacillus novalis; α-mannosidases; carbohydrate-binding module family 32
• ISSN: 2059-7983; 0907-4449; 2059-7983; 1399-0047
• DOI: 10.1107/S2059798323001663

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. The structure of the full‐length multi‐domain GH92 α‐1,2‐mannosidase from N. novalis (NnGH92) was solved and the function of the noncatalytic domains was investigated by their sequential deletion and biochemical characterization of the NnGH92 variants. This work significantly advances the structural knowledge of multi‐domain GH92 α‐1,2‐mannosidases and provides a better understanding for the future optimization of these enzymes for the degradation of yeast α‐mannan or protein glycans.