Oligo-1,6-glucosidase

 

Oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase) hydrolyses the non-reducing terminal alpha-1,6-glucosidic bonds of isomaltosaccharides, panose, and alpha-limit dextrins, but fails to act on alpha,1,4-glucosidic bonds of maltosaccharides [PMID:8370659]. The anomeric configuration of the substrate is retained, thus the mechanism proceeds via a double displacement process [PMID:9193006, PMID:10331869]. In addition, this enzyme belongs to the family 13 of glycosyl hydrolases, also known as the alpha-amylase family.

 

Reference Protein and Structure

Sequence
P21332 UniProt (3.2.1.10) IPR006047 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus cereus (Bacteria) Uniprot
PDB
1uok - CRYSTAL STRUCTURE OF B. CEREUS OLIGO-1,6-GLUCOSIDASE (2.0 Å) PDBe PDBsum 1uok
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1uok)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.10)

water
CHEBI:15377ChEBI
+
isomaltose
CHEBI:28189ChEBI
→
alpha-D-glucose
CHEBI:17925ChEBI
+
D-glucopyranose
CHEBI:4167ChEBI
Alternative enzyme names: Alpha-limit dextrinase, Dextrin 6-glucanohydrolase, Dextrin 6-alpha-glucanohydrolase, Exo-oligo-1,6-glucosidase, Isomaltase, Limit dextrinase (erroneous), Sucrase-isomaltase, Oligosaccharide alpha-1,6-glucosidase, Alpha-methylglucosidase,

Enzyme Mechanism

Introduction

The mechanism here is shown utilising isomaltose. The alpha-retaining mechanism is a double displacement process, which proceeds in two steps. In the first step, Oligo-1,6-glucosidase cleaves an alpha(1-6) glycosidic bond in its substrate, dextrin or isomaltose, and forms a covalent beta(1-6)-linked glycosyl-enzyme intermediate [PMID:10331869]. In the second step, the resulting glycosyl enzyme is hydrolysed by a water molecule [PMID:11676021]. Two active site amino acids play distinct roles in catalysis. One is the acid/base Glu 255, which protonates the glycosidic oxygen of the scissile bond in the first step, and then deprotonates the attacking water molecule in the second step. The other is the nucleophile, Asp 229, which attacks the sugar, forming the covalent linkage within the intermediate [PMID:10331869, PMID:11676021].

Catalytic Residues Roles

UniProt PDB* (1uok)
Glu255 Glu255A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, repulsive charge-charge interaction
His328 His328A Helps stabilise the reactive intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Asp199 Asp199A Acts as the catalytic nucleophile. covalently attached, nucleofuge, nucleophile, polar interaction
Asp98 Asp98A Acts to destabilise the enzyme-substrate complex ground state. repulsive charge-charge interaction, electrostatic destabiliser, steric role, electrostatic stabiliser
Asp329 Asp329A Helps to stabilise the reactive intermediates and transition states formed during the course of the reaction. transition state stabiliser
*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

bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, overall product formed, overall reactant used, intermediate formation, enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated

References

  1. Uitdehaag JC et al. (1999), Nat Struct Biol, 6, 432-436. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the alpha-amylase family. DOI:10.1038/8235. PMID:10331869.
  2. Hung VS et al. (2005), Appl Microbiol Biotechnol, 68, 757-765. alpha-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates. DOI:10.1007/s00253-005-1977-3. PMID:15940457.
  3. Watanabe K et al. (2001), Biosci Biotechnol Biochem, 65, 2058-2064. Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase. DOI:10.1271/bbb.65.2058. PMID:11676021.
  4. Watanabe K et al. (1997), J Mol Biol, 269, 142-153. The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 å resolution: structural characterization of proline-substitution sites for protein thermostabilization. DOI:10.1006/jmbi.1997.1018. PMID:9193006.
  5. Kizaki H et al. (1993), J Biochem, 113, 646-649. Polypeptide folding of Bacillus cereus ATCC7064 oligo-1,6-glucosidase revealed by 3.0 A resolution X-ray analysis. PMID:8370659.

Step 1. Asp displaces the terminal sugar in a nucleophilic substitution reaction, forming a covalent bond between the enzyme and substrate. The mechanism proceeds through a high-energy oxocarbenium-like transition state, in which the reaction centre of the sugar is planar and positively charged [PMID:10331869].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp199A polar interaction
His328A electrostatic stabiliser, hydrogen bond donor
Asp98A repulsive charge-charge interaction, electrostatic destabiliser, steric role, electrostatic stabiliser
Glu255A hydrogen bond donor
Asp329A transition state stabiliser
Asp199A nucleophile
Glu255A proton donor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, overall product formed, overall reactant used, intermediate formation

Step 2. Glu255 deprotonates the water, which initiates a nucleophilic attack on the covalently bound sugar, displacing the Asp199. The mechanism proceeds through a high-energy oxocarbenium-like transition state, in which the reaction centre of the sugar is planar and positively charged [PMID:10331869].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp199A covalently attached
His328A electrostatic stabiliser, hydrogen bond donor
Asp98A repulsive charge-charge interaction, electrostatic destabiliser, steric role, electrostatic stabiliser
Glu255A hydrogen bond acceptor, repulsive charge-charge interaction
Asp329A transition state stabiliser
Glu255A proton acceptor
Asp199A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex cleavage, overall product formed, overall reactant used, intermediate terminated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp displaces the terminal sugar in a nucleophilic substitution reaction, forming a covalent bond between the enzyme and substrate. The mechanism proceeds through a high-energy oxocarbenium-like transition state, in which the reaction centre of the sugar is planar and positively charged [PMID:10331869].

Step 2. Glu255 deprotonates the water, which initiates a nucleophilic attack on the covalently bound sugar, displacing the Asp199. The mechanism proceeds through a high-energy oxocarbenium-like transition state, in which the reaction centre of the sugar is planar and positively charged [PMID:10331869].

Products.

Contributors

Gemma L. Holliday, Christian Drew, Craig Porter, James Willey