Glycoside Hydrolase Family 164

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• Author: ^^^Zachary Armstrong^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH164
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH164.html

Figure 1. The trimeric structure of Bs164 is shown in panel A. All three protomers are shown with a surface and each chain is displayed as a cartoon diagram coloured by domain. B shows the structure of one protomer. Domain A, which has a (β/α)8 fold, is shown in green with subdomain H is shown in magenta, domain B, containing a mixed β-sheet, is shown in red and the β-sandwich of domain C is shown in blue.

Substrate specificities

The defining member of glycoside hydrolase family 164, a β-mannosidase from Bacteroidetes salyersiae (Bs164, GenbankID: EIY59668.1), was identified initially identified through rational bioinformatic selection of enzyme targets [1]. Although Bs164 was initially reported as an α-mannosidase, subsequent detailed biochemical characterization and structure determination revealed that was instead a β-mannosidase [2]. This enzyme is an exo-acting and is capable of cleaving mannooligos and β-mannosides[2].

Kinetics and Mechanism

Bs164 β-mannosidase is a retaining enzyme, as first shown by NMR [2], and follows the classical Koshland double-displacement mechanism.

Catalytic Residues

The catalytic nucleophile of Bs164 was identified as glutamate 297 through mutational analysis[2]. 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[2].

Three-dimensional structures

To date only the structure of Bs164 has been solved. Bs164 was solved using multi-wavelength anomalous diffraction of a seleno-methionine labeled protein[2]. The structure of Bs164 has been solved in an uncomplexed state and in complex with mannoimidazole, noeuromycin and 2-deoxy-2-fluoromannose. Bs164 exists as a donut shaped trimer, see 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 [3], but is previously unseen for β-mannosidases.

Family Firsts

First sterochemistry determination

Bacteroides salyersiae β-mannosidase by NMR [2]

First catalytic nucleophile identification

Bacteroides salyersiae β-mannosidase by 2-fluoromannose labeling and kinetic analysis of mutants [2]

First general acid/base residue identification

Bacteroides salyersiae β-mannosidase by kinetic analysis of mutants [2]

First 3-D structure of a GH1 enzyme

Bacteroides salyersiae β-mannosidase [2]

References

1. 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 |
2. 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 |
3. 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 |