Glycoside Hydrolase Family 92

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Glycoside Hydrolase Family GH92
Clan GH-x
Mechanism inverting
Active site residues known
CAZy DB link
http://www.cazy.org/GH92.html


Contents

Substrate specificities

The glycoside hydrolases of GH92 are exo-acting α-mannosidases. The first reported enzyme activity from this family was an α-1,2-mannosidase from Microbacterium sp. M-90 [1]. Zhu et al. reported the characterization of 22 GH92 enzymes from Bacteroides thetaiotaomicron and confirmed an exo-mode of action with α-1,2-mannosidase, α-1,3-mannosidase, α-1,4-mannosidase and α-1,6-mannosidase activities detected [2]. Tiels et al. identified a subset of GH92 enzymes, typified by CcGH92_5 from Cellulosimicrobium cellulans (formerly Arthrobacter luteus) that act to cleave yeast cell wall type mannose-1-phosphate-6-mannosides that are attached through N-linked core glycans to glycoproteins, releasing phosphate-6-mannosides (mannose-6-phosphate groups) [3]. This subfamily of mannose-1-phosphate active enzymes do not act on typical α-mannosides, which has been attributed to the replacement of the catalytic general acid (glutamic acid) with a glutamine, and other amino acids that define a phosphate binding site [3].

Kinetics and Mechanism

1H NMR studies on three GH92s that displayed α-1,2-, α-1,3- and α-1,4-mannosidase activities all generated β-mannose indicating that these enzymes catalyse glycosidic bond hydrolysis through a single displacement mechanism leading to inversion of anomeric configuration [2]. GH92 enzymes are Ca2+-dependent α-mannosidases. The requirement for the metal ion is currently restricted to only three GH families all of which are exo-α-mannosidases: GH38, GH47 and GH92. 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 achieved by a metal ion interaction with O2 and O3. Three inhibitors bound to the α-1,2-mannosidase Bt3990 in approximate 1S5/B2,5 and 1,4B/1S5 conformations indicating that catalysis is proceeds via a B2,5 transition state.

Catalytic Residues

Based on 3D structural data on the α-1,2-mannosidase Bt3990, Glu533 is the predicted general acid. This view is supported by an inactive mutant of this residue, and the conservation of the glutamate throughout the GH92 family [2]. The general base, in common with many inverting glycoside hydrolases, is more difficult to identify. Asp644 and Asp642 both lie in the canonical position expected for a general base in an inverting enzyme. Mutants of either residues inactivate 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 general base. In the case of the mannose-1-phosphate-6-mannoside cleaving α-mannosidase CcGH92_5 from Cellulosimicrobium cellulans, the enzyme lacks the typical glutamic acid general acid, which is replaced with a glutamine (Q536), which is not a proton donor [3]. The phosphate group of the substrate is a much better leaving group than a typical glycoside, and it seems likely that this does not require general acid catalysis, similar to the case seen for plant myrosinases of family GH1. As well, the replacement of the glutamic acid with glutamine may reduce charge repulsion in the active site with the anionic phosphate aglycon.

Three-dimensional structures

GH92 enzymes display a two domain structure. The small N-terminal domain is a β-sandwich and the large C-terminal domain adopts a adorned (α/α)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]. In complexes with inhibitors such as swainsonine, kifunensine, mannoimidazole, and deoxymannojirimycin the Ca2+ ion is coordinated to the 2- and 3-OH groups of the inhibitors [2, 3].

Family Firsts

First sterochemistry determination
1H NMR showed three GH92s generate β-mannose and thus these α-mannosidases are inverting enzymes [2].
First general 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 β-sandwich domain and a C-terminal adorned (α/α)6 barrel. Both domains contribute residues to the active site [2].

References

  1. Maruyama Y, Nakajima T, and Ichishima E. A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action. Carbohydr Res. 1994 Jan 3;251:89-98. PubMed ID:8149382 | HubMed [Maruyama1994]
  2. Zhu Y, Suits MD, Thompson AJ, Chavan S, Dinev Z, Dumon C, Smith N, Moremen KW, Xiang Y, Siriwardena A, Williams SJ, Gilbert HJ, and Davies GJ. Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont. Nat Chem Biol. 2010 Feb;6(2):125-32. DOI:10.1038/nchembio.278 | PubMed ID:20081828 | HubMed [Zhu2010]
  3. Tiels P, Baranova E, Piens K, De Visscher C, Pynaert G, Nerinckx W, Stout J, Fudalej F, Hulpiau P, Tännler S, Geysens S, Van Hecke A, Valevska A, Vervecken W, Remaut H, and Callewaert N. A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes. Nat Biotechnol. 2012 Dec;30(12):1225-31. DOI:10.1038/nbt.2427 | PubMed ID:23159880 | HubMed [Tiels2012]
All Medline abstracts: PubMed | HubMed
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