Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

Abstract

Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-mannosidases. Structures of Bs164 at 1.80–2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.

Synthesis of broad-specificity activity-based probes for exo-β-mannosidases

“…4 † ). 5 H260 shows a slight displacement in the complex with compound 2. We attribute this to Coulomb repulsion with the aziridine nitrogen, possibly explaining the weaker affinity of the aziridine with the Bs GH164 active site in spite of an expected higher electrophilicity.…”

“…Having candidate inhibitors 1–3 and activity-based probes 4–6 we initially tested 1, 2, and 3 against a diverse collection of well-characterised bacterial exo -β-mannosidases, including Bt Man2A, 4 Cm Man5A, 2 and Bs GH164. 5 Where covalent inhibition was observed, it was extremely slow. Activity and intact MS measurements collected following 25 hours incubation of each enzyme with 1 mM of each inhibitor under optimal conditions showed distinct patterns of reactivity for each enzyme (ESI Fig.…”

“…Bs GH164 was expressed and purified as in Armstrong and Davies. 5 Briefly, a 1 ml starter culture of pET28-His6- Bs GH164 in BL21(DE3) gold cells was used to inoculate NZYTech autoinduction media (NZYTech) containing 50 μg ml −1 of kanamycin. Expression cultures were grown at 37 °C with shaking at 250 rpm for 6 h, the temperature was then decreased to 20 °C and cultures were incubated for an additional 22 h. Expression cultures were harvested by centrifugation (5000 g , 30 min, 4 °C) and cell pellets were stored at −70 °C until purification.…”

“…1 In the human gut, exoβ-mannosidases of families GH2, GH5, and GH164 are essential for the fermentation of β-mannans, such as carob or guar endosperm (Ceratonia siliqua) galactomannan, common food additives. [2][3][4][5] In mammals, the lysosomal glycoside hydrolase family 2 (GH2 6 ) retaining exo-β-mannosidase, MANBA (EC 3.2.1.25), is responsible for the cleavage of the core Man-β-1,4-GlcNAc linkage during lysosomal turnover of N-glycoproteins. 7 Deficiency of this enzyme activity, caused by mutations in MANBA, 8,9 results in β-mannosidosis, a rare lysosomal storage disorder that can be diagnosed on the basis of elevated Manβ-1,4-GlcNAc in urine or diminished hydrolysis of 4-methylumbelliferyl (4-MU) β-D-mannopyranoside in plasma.…”

Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes

“…Additionally, the exo - β -1,4-mannosidase Bs 164 (PDB code 6T7G ( Armstrong and Davies, 2020 ) from the human gut symbiont Bacteroides salyersiae is a homotrimer with striking similarity to the GH42 β -galactosidases. Bs 164 has been classified as a member of family GH164, but regardless, the enzyme features the topology and tertiary structure of the GH42 β -galactosidases ( Figure S6 and Figure S7 ).…”