PDBsum entry 3bmw

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Transferase PDB id
Jmol PyMol
Protein chain
683 a.a. *
SO4 ×3
GOL ×6
_CA ×2
Waters ×1155
* Residue conservation analysis
PDB id:
Name: Transferase
Title: Cyclodextrin glycosyl transferase from thermoanerobacterium thermosulfurigenes em1 mutant s77p complexed with a maltohe inhibitor
Structure: Cyclomaltodextrin glucanotransferase. Chain: a. Synonym: cyclodextrin-glycosyltransferase, cgtase. Engineered: yes. Mutation: yes
Source: Thermoanaerobacterium thermosulfurigen organism_taxid: 33950. Strain: em1. Gene: amya. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.
1.60Å     R-factor:   0.145     R-free:   0.165
Authors: H.J.Rozeboom,N.Van Oosterwijk,B.W.Dijkstra
Key ref: R.M.Kelly et al. (2008). Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution. Biochem J, 413, 517-525. PubMed id: 18422488
13-Dec-07     Release date:   27-May-08    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P26827  (CDGT_THETU) -  Cyclomaltodextrin glucanotransferase
710 a.a.
683 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Cyclomaltodextrin glucanotransferase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Degrades starch to cyclodextrins by formation of a 1,4-alpha-D- glucosidic bond.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     carbohydrate metabolic process   1 term 
  Biochemical function     catalytic activity     8 terms  


Biochem J 413:517-525 (2008)
PubMed id: 18422488  
Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution.
R.M.Kelly, H.Leemhuis, H.J.Rozeboom, N.van Oosterwijk, B.W.Dijkstra, L.Dijkhuizen.
Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor towards specific transglycosylation activity.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19763564 H.Leemhuis, R.M.Kelly, and L.Dijkhuizen (2010).
Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications.
  Appl Microbiol Biotechnol, 85, 823-835.  
19180668 H.Leemhuis, R.M.Kelly, and L.Dijkhuizen (2009).
Directed evolution of enzymes: Library screening strategies.
  IUBMB Life, 61, 222-228.  
19367403 R.M.Kelly, L.Dijkhuizen, and H.Leemhuis (2009).
The evolution of cyclodextrin glucanotransferase product specificity.
  Appl Microbiol Biotechnol, 84, 119-133.  
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