Glycoside Hydrolase Family 92

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• Author: ^^^Harry Gilbert^^^
• Responsible Curator: ^^^Harry Gilbert^^^

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Glycoside Hydrolase Family GH92
Clan	none
Mechanism	inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/fam/GH92.html

Substrate specificities

GH92 enzymes are exo-acting alpha-mannosidases. The first reported enzyme activity from this family was an alpha-1,2-mannosidase from Microbacterium sp. M-90. [1] Recently the characterization of 22 GH92 enzymes from Bacteroides thetaiotaomicron confirmed an exo-mode of action with alpha-1,2-mannosidase, alpha-1,3-mannosidase, alpha-1,4-mannosidase and alpha-1,6-mannosidase activities were detected [2].

Kinetics and Mechanism

¹H-NMR studies on three GH92s that displayed alpha-1,2-, alpha-1,3- and alpha-1,4-mannosidase activities all generated beta-mannose indicating that these enzymes catalyse glycosidic bond hydrolysis through a single displacement mechanism leading to inversion of anomeric configuration [2]. GH92 enzymes are calcium-dependent alpha-mannosidases. The requirement for the metal ion is currently restricted to only three GH families all of which are exo-alpha mannosidases. Mechanistically this may indicate that the lack of distorting binding energy provided by the -2 or +1 subsites impose a requirement for conformational flexibility at the -1 subsite (recognition of the ground state and the transition state conformations), which is best achieved by a metal ion interaction with O2 and O3. Three inhibitors bound to the alpha-1,2-mannosidase Bt3990 in approximate 1S5/B2,5 and 1,4B/1S5 conformations indicate that catalysis is mediated by a B2,5 transition state.

Catalytic Residues

Based on 3D structural data on the alpha1,2-mannosidase Bt3990, Glu533 is the predicted catalytic acid. This view is supported by an inactive mutant of this residue, and the conservation of the glutamate throughout the GH92 family [2]. The catalytic base, in common with many inverting glycoside hydrolases, is more difficult to identify. Asp644 and Asp642 both lie in the canonical position one would expect for a general base in an inverting enzyme. Mutants of both residues inactivte the enzyme, however, while Asp644 is invariant, Asp642 can be an Asn or Asp in GH92 members [2]. It appears that Asp644 is the likely catalytic base.

Three-dimensional structures

GH92 enzymes display a two domain structure. The small N-terminal domain is a beta-sandwich and the large C-terminal domain adopts a adorned (alpha/alpha)₆ barrel fold. Amino acids in the active site of the enzyme, a shallow pocket, are contributed by both the N- and C-terminal domains [2].

Family Firsts

First sterochemistry determination

¹H-NMR showed three GH92s generate beta-mannose and thus these alpha-mannosidases are inverting enzymes [2].

First catalytic acid identification

Based on mutagenesis and 3D structural information the conserved catalytic acid has been identified [2].

First general base residue identification

Based on mutagenesis and 3D structural information a pair of likely catalytic bases were identified. As one of these residues is invariant, this is the proposed catalytic base [2].

First 3-D structure

The 3D structure reveals two domains; an N-terminal beta-sandwich domain and a C-terminal adorned (alpha/alpha)₆ barrel. Both domains contribute residues to the active site [2].

References

1. Maruyama Y, Nakajima T, and Ichishima E. (1994). A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action. Carbohydr Res. 1994;251:89-98. DOI:10.1016/0008-6215(94)84278-7 |

2. Zhu et al. (2010) Nature Chemical Biology in the press; DOI: 10.1038/nchembio.278 direct link.