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Glycoside Hydrolase Family 164
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|Glycoside Hydrolase Family GH164|
|Active site residues||known|
|CAZy DB link|
The defining member of glycoside hydrolase family 164, a β-mannosidase from Bacteroides salyersiae (Bs164, GenbankID: EIY59668.1), was identified initially identified through rational bioinformatic selection of enzyme targets . Although Bs164 was initially reported as an α-mannosidase, subsequent detailed biochemical characterization and structure determination revealed that was instead a β-mannosidase . This enzyme is an exo-acting and is capable of cleaving mannooligos and β-mannosides.
Kinetics and Mechanism
The catalytic nucleophile of Bs164 was identified as glutamate 297 through mutational analysis. A structural complex with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-mannopyranoside showed a covalent attachment of the inhibitor to glutamate 297, reinforcing the assignment of Glu297 as the catalytic nucleophile. The acid/base residue, Glu160 is positioned to perform anti-protonation of the leaving group, typical of clan GH-A glycoside hydrolases. This residue forms hydrogen bonding interactions with both the endocyclic nitrogen in noeuromycin and imidazole nitrogen in mannoimidazole. Complete loss of activity by the E160Q variant confirmed the assignment of Glu160 as the acid/base residue.
The structure of Bs164 was solved using multi-wavelength anomalous diffraction of a seleno-methionine labeled protein . The structure of Bs164 has been solved in an uncomplexed state (PDB ID 6T5O), in complex with mannoimidazole (PDB ID 6T7G) and noeuromycin (PDB ID 6T6G), and as a covalent 2-deoxy-2-fluoromannosyl-enzyme intermediate (PDB ID 6T75). Bs164 exists as a donut shaped trimer (Figure 1A). Each trimer-donut has an outer diameter of approximately 100 Å and an internal diameter of between 30 and 35 Å. The individual Bs164 chains contain three clearly defined domains: a modified (β/α)8 barrel, a domain containing a seven membered mixed β-sheet sandwiched between α-helices, and a β-sheet domain (Figure 1B). The catalytic residues are present on strands 4 (acid/base) and 7 (nucleophile) of the (β/α)8 barrel indicating that GH164 belongs to clan GH-A glycoside hydrolases. This domain architecture is quite similar to that seen for family GH42 enzymes , but is previously unseen for β-mannosidases.
- First sterochemistry determination
- Bacteroides salyersiae β-mannosidase by NMR 
- First catalytic nucleophile identification
- Bacteroides salyersiae β-mannosidase by 2-fluoromannose labeling and kinetic analysis of mutants 
- First general acid/base residue identification
- Bacteroides salyersiae β-mannosidase by kinetic analysis of mutants 
- First 3-D structure of a GH1 enzyme
- Bacteroides salyersiae β-mannosidase 
- Helbert W, Poulet L, Drouillard S, Mathieu S, Loiodice M, Couturier M, Lombard V, Terrapon N, Turchetto J, Vincentelli R, and Henrissat B. (2019). Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space. Proc Natl Acad Sci U S A. 2019;116(13):6063-6068. DOI:10.1073/pnas.1815791116 |
- Armstrong Z and Davies GJ. (2020). Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164. J Biol Chem. 2020;295(13):4316-4326. DOI:10.1074/jbc.RA119.011591 |
- Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, and Wakagi T. (2002). Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol. 2002;322(1):79-91. DOI:10.1016/s0022-2836(02)00746-5 |