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Difference between revisions of "Glycoside Hydrolase Family 92"

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* [[Author]]: [[User:Harry Gilbert|Harry Gilbert]], [[User:Spencer Williams|Spencer Williams]]
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|'''Active site residues'''
 
|'''Active site residues'''
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|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 
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| colspan="2" |http://www.cazy.org/fam/GH92.html
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== Substrate specificities ==
 
== Substrate specificities ==
GH92 enzymes are exo-acting alpha-mannosidases. The first reported enzyme activity from this family was an alpha1,2-mannosidase from ''Microbacterium'' sp. M-90. <cite>#1</cite> Recently the characterization of 22 GH92 enzymes from ''Bacteroides thetaiotaomicron'' confirmed an exo-mode of action with alpha1,2-mannosidase, alpha1,3-mannosidase, alpha1,4-mannosidase and alpha1,6-mannosidase activities were detected <cite>#2</cite>.
+
The [[glycoside hydrolases]] of GH92 are [[exo]]-acting &alpha;-mannosidases. The first reported enzyme activity from this family was an &alpha;-1,2-mannosidase from ''Microbacterium'' sp. M-90 <cite>Maruyama1994</cite>. Zhu ''et al.'' reported the characterization of 22 GH92 enzymes from ''Bacteroides thetaiotaomicron'' and 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 detected <cite>Zhu2010</cite>. 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) <cite>Tiels2012</cite>. This subfamily of mannose-1-phosphate active enzymes do not act on typical &alpha;-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 <cite>Tiels2012</cite>.
  
 
== Kinetics and Mechanism ==
 
== Kinetics and Mechanism ==
1H-NMR studies on three GH92s that displayed alpha1,2-, alpha1,3- and alpha1,4-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 <cite>Zhu et al</cite>. 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 alpha1,2-mannosidase Bt3990 in approximate 1S5/B2,5 and 1,4B/1S5 conformations indicate that catalysis is mediated by a Boat2,5 transition state.
+
<sup>1</sup>H 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 [[inverting|inversion]] of anomeric configuration <cite>Zhu2010</cite>. GH92 enzymes are Ca<sup>2+</sup>-dependent &alpha;-mannosidases. The requirement for the metal ion is currently restricted to only three GH families all of which are [[exo]]-&alpha;-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 &alpha;-1,2-mannosidase Bt3990 in approximate <sup>1</sup>''S''<sub>5</sub>/''B''<sub>2,5</sub> and <sup>1,4</sup>''B''/<sup>1</sup>''S''<sub>5</sub> conformations indicating that catalysis is proceeds via a ''B''<sub>2,5</sub> [[transition state]].
  
 
== Catalytic Residues ==
 
== 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. 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. It appears that Asp644 is the likely catalytic base.
+
Based on 3D structural data on the &alpha;-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 <cite>Zhu2010</cite>. 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 <cite>Zhu2010</cite>. It appears that Asp644 is the likely catalytic [[general base]]. In the case of the mannose-1-phosphate-6-mannoside cleaving &alpha;-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 <cite>Tiels2012</cite>. 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 ==
 
== 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.
+
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;)<sub>6</sub> barrel fold. Amino acids in the active site of the enzyme, a shallow pocket, are contributed by both the N- and C-terminal domains <cite>Zhu2010</cite>. In complexes with inhibitors such as swainsonine, kifunensine, mannoimidazole, and deoxymannojirimycin the Ca<sup>2+</sup> ion is coordinated to the 2- and 3-OH groups of the inhibitors <cite>Zhu2010 Tiels2012</cite>.
  
 
== Family Firsts ==
 
== Family Firsts ==
;First sterochemistry determination: 1H-NMR showed three GH92s generate beta-mannose and thus these alpha-mannosidases are inverting enzymes.
+
;First sterochemistry determination: <sup>1</sup>H NMR showed three GH92s generate &beta;-mannose and thus these &alpha;-mannosidases are [[inverting]] enzymes <cite>Zhu2010</cite>.
;First catalytic acid identification: Based on mutagenesis and 3D structural information the conserved catalytic acid has been identified.
+
;First [[general acid]] identification: Based on mutagenesis and 3D structural information the conserved catalytic acid has been identified <cite>Zhu2010</cite>.
;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.  
+
;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 <cite>Zhu2010</cite>.  
;First 3-D structure: The 3D structure reveals two domains; an N-terminal beta-sandwich domain and a C-terminal adorned (alha/alpha)6 barrel. Both domains contribute residues to the active site.
+
;First 3-D structure: The 3D structure reveals two domains; an N-terminal &beta;-sandwich domain and a C-terminal adorned (&alpha;/&alpha;)<sub>6</sub> barrel. Both domains contribute residues to the active site <cite>Zhu2010</cite>.
  
 
== References ==
 
== References ==
 
<biblio>
 
<biblio>
#1 pmid=8149382
+
#Maruyama1994 pmid=8149382
#2 Zhu
+
#Zhu2010 pmid=20081828
 +
#Tiels2012 pmid=23159880
 
</biblio>
 
</biblio>
  
  
[[Category:Glycoside Hydrolase Families|GH92]]
+
[[Category:Glycoside Hydrolase Families|GH092]]

Latest revision as of 14:19, 18 December 2021

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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


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. (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 | PubMed ID:8149382 [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. (2010). Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont. Nat Chem Biol. 2010;6(2):125-32. DOI:10.1038/nchembio.278 | PubMed ID:20081828 [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. (2012). A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes. Nat Biotechnol. 2012;30(12):1225-31. DOI:10.1038/nbt.2427 | PubMed ID:23159880 [Tiels2012]

All Medline abstracts: PubMed