Cyclomaltodextrin glucanotransferase

 

Cyclodextrin glycosyltransferases (CGTases) are industrially important enzymes that produce cyclodextrins from starch by intramolecular transglycosylation. They belong to the important alpha-amylase family (family 13) of glycosyl hydrolases and catalyse the cyclisation of part of a (1->4)-alpha-D-glucan chain by the formation of a (1->4)-alpha-D-glucosidic bond. This enzyme also acts as a hydrolase or a transglycosylase, depending on the identity of the acceptor molecule in the final stages of the reaction.

 

Reference Protein and Structure

Sequence
P43379 UniProt (2.4.1.19) IPR006047 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus circulans (Bacteria) Uniprot
PDB
1cdg - NUCLEOTIDE SEQUENCE AND X-RAY STRUCTURE OF CYCLODEXTRIN GLYCOSYLTRANSFERASE FROM BACILLUS CIRCULANS STRAIN 251 IN A MALTOSE-DEPENDENT CRYSTAL FORM (2.0 Å) PDBe PDBsum 1cdg
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1cdg)
Cofactors
Calcium(2+) (3)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.19)

Celloheptaose
CHEBI:3524ChEBI
→
alpha-cyclodextrin
CHEBI:40585ChEBI
+
beta-D-glucose
CHEBI:15903ChEBI
Alternative enzyme names: Alpha-1,4-glucan 4-glycosyltransferase, cyclizing, Alpha-cyclodextrin glucanotransferase, Alpha-cyclodextrin glycosyltransferase, Beta-cyclodextrin glycosyltransferase, Gamma-cyclodextrin glycosyltransferase, Bacillus macerans amylase, BMA, CGTase, Cyclodextrin glucanotransferase, Cyclodextrin glycosyltransferase, Cyclomaltodextrin glucotransferase, Cyclomaltodextrin glycosyltransferase, Konchizaimu, Neutral-cyclodextrin glycosyltransferase, 1,4-alpha-D-glucan 4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing),

Enzyme Mechanism

Introduction

This enzyme has an Asp-Asp-Glu catalytic triad that is absolutely conserved in all the amylolytic enzymes.This triad occurs in the so-called domain A of the protein.

The first step of the cyclisation reaction is the binding of the starch chain at binding site 1 (in the E-domain, which includes part of the Ca3 binding site). After this event, binding is extended to the active site through a secondary binding site (also in the E domain) which is located near a groove leading to the active site.

Cleavage of the starch chain is initiated by the nucleophilic attack of Asp229 at one of the alpha(1-->4) glycosidic bonds (breaking the bond) leading to a beta(1-->4) glycosidically linked covalent intermediate. In the cyclase reaction, the non-reducing end of the covalently linked intermediate then migrates to the acceptor site. This acceptor then attacks the −1 glucose C1 atom leading to a cyclodextrin product (cyclisation).

If the acceptor is a water molecule, the the linear hydrolysis product is formed (hydrolysis).

If the acceptor molecule is another linear maltooligosaccharide then the product is a longer linear oligosaccharide (transglycosylation or disproportionation).

Catalytic Residues Roles

UniProt PDB* (1cdg)
Asp355 Asp328A Negatively charged side chain also stabilises the oxocarbenium transition states hydrogen bond acceptor, electrostatic stabiliser
Asp256 Asp229A Acts as a nucleophile to attack the sugar, forming the covalent linkage within the intermediate. Its carboxylate group carbonyl oxygen also stabilises the transition state. nucleofuge, nucleophile
Glu284 Glu257A Acts as a base protonates the glycosidic oxygen of the scissile bond in the first step and then deprotonates the attacking hydroxyl group in the second step. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg254, His354 Arg227A, His327A Helps stabilise the intermediates formed. hydrogen bond donor, electrostatic 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, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, overall product formed, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated, intermediate collapse

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. Li Z et al. (2016), Int J Biol Macromol, 83, 111-116. Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans. DOI:10.1016/j.ijbiomac.2015.11.042. PMID:26608005.
  3. Ban X et al. (2015), Int J Biol Macromol, 76, 224-229. Mutations at calcium binding site III in cyclodextrin glycosyltransferase improve β-cyclodextrin specificity. DOI:10.1016/j.ijbiomac.2015.02.036. PMID:25748847.
  4. Kumar V (2010), Carbohydr Res, 345, 893-898. Analysis of the key active subsites of glycoside hydrolase 13 family members. DOI:10.1016/j.carres.2010.02.007. PMID:20227065.
  5. van der Veen BA et al. (2000), Eur J Biochem, 267, 658-665. The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms. DOI:10.1046/j.1432-1327.2000.01031.x.
  6. Uitdehaag JCM et al. (2000), Biochemistry, 39, 7772-7780. Structures of Maltohexaose and Maltoheptaose Bound at the Donor Sites of Cyclodextrin Glycosyltransferase Give Insight into the Mechanisms of Transglycosylation Activity and Cyclodextrin Size Specificity†,‡. DOI:10.1021/bi000340x.
  7. Mosi R et al. (1997), Biochemistry, 36, 9927-9934. Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme†. DOI:10.1021/bi970618u. PMID:9245426.
  8. Knegtel RM et al. (1995), J Biol Chem, 270, 29256-29264. Crystallographic Studies of the Interaction of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251 with Natural Substrates and Products. DOI:10.1074/jbc.270.49.29256. PMID:7493956.
  9. Lawson CL et al. (1994), J Mol Biol, 236, 590-600. Nucleotide Sequence and X-ray Structure of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251 in a Maltose-dependent Crystal Form. DOI:10.1006/jmbi.1994.1168. PMID:8107143.

Step 1. Asp229 initiates a nucleophilic attack upon the carbon of the ether linkage between the two sugar rings in a substitution reaction, which eliminates a free sugar molecule, with concomitant deprotonation of Glu257.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg227A hydrogen bond donor, electrostatic stabiliser
His327A hydrogen bond donor, electrostatic stabiliser
Asp328A hydrogen bond acceptor, electrostatic stabiliser
Glu257A hydrogen bond donor
Glu257A proton donor
Asp229A nucleophile

Chemical Components

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

Step 2. Glu257 deprotonates a the acceptor OH, which initiates a nucleophilic attack upon the covalently bound sugar in a substitution reaction, eliminating Asp229.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg227A hydrogen bond donor, electrostatic stabiliser
His327A hydrogen bond donor, electrostatic stabiliser
Asp328A hydrogen bond acceptor, electrostatic stabiliser
Glu257A hydrogen bond acceptor, proton acceptor
Asp229A nucleofuge

Chemical Components

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

Step 1. Asp229 initiates a nucleophilic attack upon the carbon of the ether linkage between the two sugar rings in a substitution reaction, which eliminates a free sugar molecule, with concomitant deprotonation of Glu257.

Step 2. Glu257 deprotonates a the acceptor OH, which initiates a nucleophilic attack upon the covalently bound sugar in a substitution reaction, eliminating Asp229.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Nozomi Nagano, Craig Porter