Mannosyl-oligosaccharide 1,2-alpha-mannosidase

 

Class I alpha-1,2-mannosidases are conserved throughout eukaryotic evolution and are members of the glycoside hydrolase family 47. They regulate the maturation of N-glycans during glycoprotein biosynthesis. N-glycan formation begins with the transfer of a preformed oligosaccharide precursor, usually Glc3Man9ClcNac2, to nascent polypeptide chains. The oligosaccharide precursor is then trimmed immediately by alpha-glucosidases and alpha-mannosidases in the endoplasmic reticulum or the golgi apparatus. Besides their importance in N-glycan maturation, endoplasmic reticulum and golgi apparatus processing glycosidases and mannosidases also play a role in protein folding "quality control". Trimming of mannose residues in the endoplasmic reticulum acts as a signal to target misfolded glycoproteins for degradation by the proteasome, which ensures only correctly folded proteins are transported to their final destination.

Mannosyl-oligosaccharide 1,2-alpha-mannosidase is the only alpha-mannosidase in Saccharomyces cerevisae and it removes a single mannose residue from Man(9)(GlcNAc)(2) to form Man(8)(GlcNAc)(2) in the endoplasmic reticulum as do equivalent enzymes in higher organisms. Class I enzymes found in the golgi apparatus remove all four linked alpha-mannose residues.

 

Reference Protein and Structure

Sequence
Q9UKM7 UniProt (3.2.1.113) IPR001382 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1x9d - Crystal Structure Of Human Class I alpha-1,2-Mannosidase In Complex With Thio-Disaccharide Substrate Analogue (1.41 Å) PDBe PDBsum 1x9d
Catalytic CATH Domains
1.50.10.10 CATHdb (see all for 1x9d)
Cofactors
Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.2.1.113)

alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-alpha-D-Man-(1->6)]-alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc
CHEBI:59579ChEBI
+
water
CHEBI:15377ChEBI
→
beta-D-mannose
CHEBI:28563ChEBI
+
alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)-[alpha-D-Man-(1->2)-alpha-D-Man-(1->3)]-alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc
CHEBI:64052ChEBI
Alternative enzyme names: 1,2-alpha-mannosidase, Man9-mannosidase, Exo-alpha-1,2-mannanase, Glycoprotein processing mannosidase I, Mannose-9 processing alpha-mannosidase, Mannosidase 1A, Mannosidase 1B, Mannosidase I, ManI, 1,2-alpha-mannosyl-oligosaccharide alpha-D-mannohydrolase,

Enzyme Mechanism

Introduction

This computationally derived mechanism consists of five steps in which Asp463 transfers its proton to water; this "activated" water then protonates the mannose (which is held in an ALPH-compliant 3S1-like conformation within the active site) leaving group. Glu599 activates the nucleophilic water, the water attacks and causes an inversion of stereochemistry at the actibde centre. Glu599 then deprotonates the nucleophile to generate the final products. The products subsequently leave the enzyme, and the liberated d-mannose molecule will flip into the ground-state 4C1 chair in an independent conformational process. Finally, a proton exchange between Glu599 and Asp463 will reset the enzyme for a next catalytic cycle.

Catalytic Residues Roles

UniProt PDB* (1x9d)
Glu330, Arg334 Glu330(169)A, Arg334(173)A Specifically orients Water8 for electrostatic transition-state stabilisation of the glycon ring oxygen atom. steric role
Asp463, Glu599 Asp463(302)A, Glu599(438)A Acts as a general acid/base in various steps throughout the reaction. proton acceptor, electrostatic stabiliser, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution, native state of enzyme regenerated

References

  1. Cantú D et al. (2008), Carbohydr Res, 343, 2235-2242. Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum α-(1→2)-mannosidase I. DOI:10.1016/j.carres.2008.05.026. PMID:18619586.
  2. Mulakala C et al. (2002), Proteins, 49, 125-134. Understanding protein structure-function relationships in Family 47 ?-1,2-mannosidases through computational docking of ligands. DOI:10.1002/prot.10206. PMID:12211022.
  3. Vallee F et al. (2000), J Biol Chem, 275, 41287-41298. Structural Basis for Catalysis and Inhibition ofN-Glycan Processing Class I  1,2-Mannosidases. DOI:10.1074/jbc.m006927200. PMID:10995765.

Step 1. Asp463 is deprotonated by an active site water molecule, activating the water molecule for the next step.

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Catalytic Residues Roles

Residue Roles
Asp463(302)A proton donor

Chemical Components

proton transfer

Step 2. The leaving group is protonated by the activated water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp463(302)A electrostatic stabiliser
Glu330(169)A steric role
Arg334(173)A steric role

Chemical Components

proton transfer

Step 3. Glu559 activates the nucleophilic water, which attacks the substrate with an inversion of stereochemistry at the active site.

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Catalytic Residues Roles

Residue Roles
Glu599(438)A increase nucleophilicity
Glu330(169)A steric role
Arg334(173)A steric role

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 4. Glu599 deprotonates the water, returning the product to a neutral state.

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Catalytic Residues Roles

Residue Roles
Glu599(438)A proton acceptor

Chemical Components

proton transfer

Step 5. A proton is transferred from Glu599 to Asp463 to return the enzyme to its ground state for the next catalytic cycle.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp463(302)A proton acceptor
Glu599(438)A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp463 is deprotonated by an active site water molecule, activating the water molecule for the next step.

Step 2. The leaving group is protonated by the activated water molecule.

Step 3. Glu559 activates the nucleophilic water, which attacks the substrate with an inversion of stereochemistry at the active site.

Step 4. Glu599 deprotonates the water, returning the product to a neutral state.

Step 5. A proton is transferred from Glu599 to Asp463 to return the enzyme to its ground state for the next catalytic cycle.

Products.

Introduction

The catalytic acidic residues and the calcium ion required for activity are located in the centre of an (alpha-alpha)7 barrel at the top of a beta-hairpin. The enzyme is an inverting hydrolase, causing an inversion of configuration at C1 of the tenth oligosaccharide residue.

The reaction proceeds as follows: Glu132 acts as a catalytic base and abstracts a proton from water. A hydrogen bond with Arg136 increases the acidity of Glu132. Asp275 is then thought to act as the catalytic acid donating a proton to the leaving group.

Catalytic Residues Roles

UniProt PDB* (1x9d)
Glu132 Glu132(99)A Acts as a general acid/base, it starts the reaction in a neutral state, and furnishes the alcohol leaving group with its proton. It is returned to its initial protonation step by water in an inferred return step. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp275 Asp275(242)A Acts as a general acid/base, abstracting a proton from the nucleophilic water molecule. It is reprotonated from another water molecule in an inferred return step. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg136 Arg136(103)A Stabilises Glu132 in its neutral state. activator, hydrogen bond donor, electrostatic stabiliser
Glu435 Glu435(397)A Activates water. activator
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution, atom stereo change, overall reactant used, overall product formed, proton relay, rate-determining step, native state of enzyme regenerated, inferred reaction step

References

  1. Vallee F et al. (2000), J Biol Chem, 275, 41287-41298. Structural Basis for Catalysis and Inhibition ofN-Glycan Processing Class I  1,2-Mannosidases. DOI:10.1074/jbc.m006927200. PMID:10995765.
  2. Cantú D et al. (2008), Carbohydr Res, 343, 2235-2242. Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum α-(1→2)-mannosidase I. DOI:10.1016/j.carres.2008.05.026. PMID:18619586.
  3. Karaveg K et al. (2005), J Biol Chem, 280, 16197-16207. Mechanism of Class 1 (Glycosylhydrolase Family 47)  -Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control. DOI:10.1074/jbc.m500119200. PMID:15713668.
  4. Tatara Y et al. (2003), J Biol Chem, 278, 25289-25294. Identification of Catalytic Residues of Ca2+-independent 1,2- -D-Mannosidase from Aspergillus saitoi by Site-directed Mutagenesis. DOI:10.1074/jbc.m302621200. PMID:12702721.
  5. Mulakala C et al. (2002), Proteins, 49, 125-134. Understanding protein structure-function relationships in Family 47 ?-1,2-mannosidases through computational docking of ligands. DOI:10.1002/prot.10206. PMID:12211022.
  6. Jordan IK et al. (2001), Bioinformatics, 17, 965-976. Sequence and structural aspects of functional diversification in class I alpha-mannosidase evolution. PMID:11673242.
  7. Tremblay LO et al. (1999), Glycobiology, 9, 1073-1078. Cloning and expression of a specific human alpha 1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis. PMID:10521544.

Step 1. Asp275 deprotonates water, which initiates a nucleophilic attack on the anomeric carbon, displacing the OR group in a substitution. The released alcohol deprotonates a second water molecule, which deprotonates Glu132 in turn.

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Catalytic Residues Roles

Residue Roles
Arg136(103)A hydrogen bond donor, activator
Asp275(242)A hydrogen bond acceptor
Glu132(99)A hydrogen bond donor
Glu435(397)A activator
Asp275(242)A proton acceptor
Glu132(99)A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, atom stereo change, overall reactant used, overall product formed, proton relay, rate-determining step

Step 2. In an inferred step, Asp275 and Glu132 are returned to their starting states through a proton relay chain of water molecules.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp275(242)A hydrogen bond donor
Glu132(99)A hydrogen bond acceptor
Arg136(103)A hydrogen bond donor, electrostatic stabiliser
Glu435(397)A activator
Glu132(99)A proton acceptor
Asp275(242)A proton donor

Chemical Components

proton transfer, proton relay, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp275 deprotonates water, which initiates a nucleophilic attack on the anomeric carbon, displacing the OR group in a substitution. The released alcohol deprotonates a second water molecule, which deprotonates Glu132 in turn.

Step 2. In an inferred step, Asp275 and Glu132 are returned to their starting states through a proton relay chain of water molecules.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Sophie T. Williams, James W. Murray, Craig Porter, Katherine Ferris