This is a repository copy of Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/155946/ Version: Accepted Version Article: Armstrong, Zachary and Davies, Gideon J orcid.org/0000-0002-7343-776X (2020) Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164. The Journal of biological chemistry. pp. 4316-4326. ISSN 1083-351X https://doi.org/10.1074/jbc.RA119.011591 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ JBC Papers in Press. Published on December 22, 2019 as Manuscript RA119.011591 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.011591 Structure and Function of Bs164 Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164. Authors: Zachary Armstrong1, Gideon J. Davies1* From the 1Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, United Kingdom Running Title: Structure and Function of Bs164 * To whom correspondence should be addressed: Gideon J. Davies: Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, United Kingdom; [email protected]; Tel. (+44) 01904 328260 Keywords: Mannan, Mannose, Glycoside Hydrolase, Microbiome, β-Mannosidase, Gut Microbe, Symbiont, GH164, Carbohydrate Active Enzyme Downloaded from ABSTRACT of the mechanisms commensal microbes use for Recent work exploring protein sequence nutrient acquisition. http://www.jbc.org/ space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. Introduction GH164 genes are present in several commensal Mannose is an essential component of bacteria, implicating these genes in the protein human N-glycans (1), storage polymers – degradation of dietary glycans. However, little is mannan and glucomannan in plants (2) and at UNIVERSITY OF YORK on January 22, 2020 known about the structure, mechanism of action mannogen in Leishmania (3) – and has even been and substrate specificity of these enzymes. Herein observed to play a role in enzyme substrate we report the biochemical characterization and recognition (4). Considering the roles of mannose crystal structures of the founding member of this containing polymers, it is no surprise that nature family (Bs164) from the human gut symbiont has developed a variety of enzymes to either Bacteroides salyersiae. Previous reports of this modify the properties of these polymers or release enzyme indicated that it has α-mannosidase their stored energy. The majority of enzymes that activity, however we conclusively show that it degrade mannose polymers are classified as cleaves only β-mannose linkages. Using NMR glycoside hydrolases (GHs). The Carbohydrate spectroscopy, detailed enzyme kinetics of wild- Active Enzymes (CAZy) database (5) type and mutant Bs164, and multi-angle light (http://www.cazy.org) categorizes all known GHs scattering we found that it is a trimeric retaining into > 160 families, including several with known β-mannosidase, that is susceptible to several mannosidase activity. A major focus of recent known mannosidase inhibitors. X-ray study has centred on how the enteric bacteria crystallography revealed the structure of Bs164 – employ GHs to degrade variety of the first known structure of a GH164 – at 1.91 Å polysaccharides including the mannose polymers resolution. Bs164 is composed of three domains: present in the human gut (6-10), resulting in a a (β/α)8 barrel, a trimerization domain and a β- better understanding of degradation mechanisms sandwich domain, representing a previously that underpin the nutrient acquisition by unobserved structural fold for β-mannosidases. commensal bacteria. Structures of Bs164 at 1.80-2.55 Å resolution in Recent work by Helbert and co-workers complex with the inhibitors noeuromycin, (11) identified a new family of mannosidases: mannoimidazole or DNP 2-deoxy-2-fluoro- family GH164. This was accomplished through mannose reveal the residues essential for the combination of sequence space exploration, specificity and catalysis including the catalytic gene synthesis and high-throughput activity nucleophile (Glu297) and acid/base residue assays. The founding member of GH164 – which (Glu160). These findings further our knowledge we shall herein refer to as Bs164 – originates from the enteric bacterium Bacteroides salyersiae 1 Structure and Function of Bs164 CL02T12C01 and was initially reported by the both endo and exo-acting GH families. The authors to have α-mannosidase activity. This presence of GH5, a family that contains endo-β- family at present contains seventeen genes, the mannanases, and GH2, a family containing both majority of which belong to host associated β-galactosidases and β-mannosidases, and the strains. However, due to the preliminary nature of absence of α-mannose targeting families suggests this discovery very little is known about the that these operons function to degrade dietary β- structure, action mechanism or substrate mannan, glucomannan or galactomannan rather specificity of GH164 enzymes. than N-glycans. The B. salyersiae genome also Here we present the detailed biochemical contains both GH5 and GH2 encoding genes and structural analysis of Bs164. In contrast to suggesting that it may also target mannans, initial reports, this enzyme has no α-mannosidase though with enzymes from separate loci. activity; instead, Bs164 cleaves β-mannosidic linkages in aryl b-mannosidase and Bs164 is a retaining β-mannosidase mannooligosaccharides. X-ray crystal structures The Bs164 protein containing an N- of Bs164 reveal a homo-trimeric quaternary terminal hexahistidine tag was purified using structure with each individual chain containing immobilized metal affinity chromatography three domains. The catalytic domain of Bs164 followed by size exclusion chromatography. The Downloaded from consists of a (β/α)8 barrel with catalytic residues hexahistidine tag was then removed using 3C on β-strands 4 and 7 placing this family in clan protease, and the untagged protein was further GH-A. NMR analysis revealed a retaining purified using an additional size exclusion mechanism and site directed mutagenesis chromatography step. Bs164 eluted from the size http://www.jbc.org/ confirmed the assignment of the nucleophile and exclusion earlier than would be expected for a acid/base catalytic residues. Structures of 74.4 kDa protein. To determine whether Bs164 inhibitor complexes show the conserved catalytic exists as a multimer in solution we subjected machinery involved in substrate recognition and purified Bs164 to SEC-MALS (size exclusion catalysis and provide insight into the likely chromatography with multi-angle light at UNIVERSITY OF YORK on January 22, 2020 conformational itinerary for mannoside scattering). A single peak was observed with a hydrolysis. Taken together, this work provides a calculated molecular mass of 227 kDa, signifying thorough biochemical basis for the β-mannoside that Bs164 forms trimers in solution. hydrolysis catalyzed by GH164 enzymes. To assess the substrate tolerance of Bs164 we assayed the purified enzyme with a variety of Results and Discussion synthetic aryl glycosides and Operonic context of GH164s mannooligosaccharides (see methods for The GH164 genes present in the CAZy complete list). Bs164 shows no activity towards database are confined to the Bacteroidetes (5), a D-xylose, D- or L-arabinose, D-galactose, D- phylum that is well known for carbohydrate glucose, or L-fucose containing substrates. utilization operons (12). In addition to the GH164 Furthermore no activity was observed towards α- from B. saylersiae four genes originate from the D-linked mannosides, contradicting the initial Alistipes genus, commonly found in the human report of enzyme activity by Helbert et al (11). gut (13) , ten from the Capnocytophaga, typically Bs164 did, however, have significant activity found in the oralpharengyl tract (14), and one against β-linked aryl mannosides with a pH each belonging to the marine bacterium optimum 5.5 (Figure 2). This pH was used in Flammeovirga and the lichen associated assays to characterize the specificity if this Mucilaginibacter. Examination of the operonic enzyme towards β-mannoside hydrolysis (Table context of the GH164 gives some clue to the 1). The specificity constant for pNP β-man polysaccharides which they target (Figure 1). The hydrolysis was approximately 6 times that seen locus surrounding Bs164 is limited to two other for MU β-man which we attribute to pNP being a genes, a hybrid two component sensor (HTCS) slightly better leaving group. This enzyme is also and an arylsulfatase like protein, however the able to