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PDBsum entry 1dtu

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protein ligands metals links
Transferase PDB id
1dtu
Jmol
Contents
Protein chain
686 a.a. *
Ligands
GLC-GLC
GLC-G6D
GLC-GLC-GLC ×2
BGC-GLC-GLC-GLC-
GLC
ADH
Metals
_CA ×2
Waters ×273
* Residue conservation analysis
PDB id:
1dtu
Name: Transferase
Title: Bacillus circulans strain 251 cyclodextrin glycosyltransferase: a mutant y89d/s146p complexed to an hexasaccharide inhibitor
Structure: Protein (cyclodextrin glycosyltransferase). Chain: a. Engineered: yes. Mutation: yes
Source: Bacillus circulans. Organism_taxid: 1397. Strain: 251. Cellular_location: extracellular. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.
Resolution:
2.40Å     R-factor:   0.209     R-free:   0.248
Authors: J.C.M.Uitdehaag,K.H.Kalk,B.W.Dijkstra
Key ref:
B.A.van der Veen et al. (2000). Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production. J Mol Biol, 296, 1027-1038. PubMed id: 10686101 DOI: 10.1006/jmbi.2000.3528
Date:
13-Jan-00     Release date:   06-Mar-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P43379  (CDGT2_BACCI) -  Cyclomaltodextrin glucanotransferase
Seq:
Struc:
 
Seq:
Struc:
713 a.a.
686 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.2.4.1.19  - 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  

 

 
DOI no: 10.1006/jmbi.2000.3528 J Mol Biol 296:1027-1038 (2000)
PubMed id: 10686101  
 
 
Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.
B.A.van der Veen, J.C.Uitdehaag, D.Penninga, G.J.van Alebeek, L.M.Smith, B.W.Dijkstra, L.Dijkhuizen.
 
  ABSTRACT  
 
Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Schematic representation of the CGTase catalyzed reactions. The circles represent glucose residues; the white circles indicate the reducing end sugars. (a) Cyclization, (b) coupling, (c) disproportionation, (d) hydrolysis
Figure 2.
Figure 2. Schematic representation of the hydrogen bonds between the B. circulans strain 251 CGTase and a maltononaose inhibitor bound at the active site. In this work the subsites will be numbered according to the general subsite labeling scheme recently proposed for all glycosyl hydrolases [Davies et al 1997], in which the glycosidic bond between -1 and +I is the scissile bond, and the substrate reducing end is at position +2. This scheme is the inverse of that used in earlier work of our groups.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 296, 1027-1038) copyright 2000.  
  Figures were selected by an automated process.  

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.  
  21048872 P.Fernandes (2010).
Enzymes in food processing: a condensed overview on strategies for better biocatalysts.
  Enzyme Res, 2010, 862537.  
19367403 R.M.Kelly, L.Dijkhuizen, and H.Leemhuis (2009).
The evolution of cyclodextrin glucanotransferase product specificity.
  Appl Microbiol Biotechnol, 84, 119-133.  
19190904 Z.Li, J.Zhang, M.Wang, Z.Gu, G.Du, J.Li, J.Wu, and J.Chen (2009).
Mutations at subsite -3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing alpha-cyclodextrin specificity.
  Appl Microbiol Biotechnol, 83, 483-490.  
18421582 H.F.Alves-Prado, A.A.Carneiro, F.C.Pavezzi, E.Gomes, M.Boscolo, C.M.Franco, and R.da Silva (2008).
Production of cyclodextrins by CGTase from Bacillus clausii using different starches as substrates.
  Appl Biochem Biotechnol, 146, 3.  
18283101 R.M.Kelly, H.Leemhuis, L.Gätjen, and L.Dijkhuizen (2008).
Evolution toward small molecule inhibitor resistance affects native enzyme function and stability, generating acarbose-insensitive cyclodextrin glucanotransferase variants.
  J Biol Chem, 283, 10727-10734.  
16204493 K.Fujii, H.Minagawa, Y.Terada, T.Takaha, T.Kuriki, J.Shimada, and H.Kaneko (2005).
Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses.
  Appl Environ Microbiol, 71, 5823-5827.  
15630515 Q.Qi, and W.Zimmermann (2005).
Cyclodextrin glucanotransferase: from gene to applications.
  Appl Microbiol Biotechnol, 66, 475-485.  
14705029 H.Leemhuis, H.J.Rozeboom, B.W.Dijkstra, and L.Dijkhuizen (2004).
Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge.
  Proteins, 54, 128-134.
PDB code: 1pj9
12492486 H.Leemhuis, B.W.Dijkstra, and L.Dijkhuizen (2003).
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.
  Eur J Biochem, 270, 155-162.  
11856334 T.P.Frandsen, M.M.Palcic, and B.Svensson (2002).
Substrate recognition by three family 13 yeast alpha-glucosidases.
  Eur J Biochem, 269, 728-734.  
11257505 E.A.MacGregor, S.Janecek, and B.Svensson (2001).
Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.
  Biochim Biophys Acta, 1546, 1.  
11288183 J.C.Uitdehaag, B.A.van der Veen, L.Dijkhuizen, R.Elber, and B.W.Dijkstra (2001).
Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase.
  Proteins, 43, 327-335.  
11282590 Y.Terada, H.Sanbe, T.Takaha, S.Kitahata, K.Koizumi, and S.Okada (2001).
Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases.
  Appl Environ Microbiol, 67, 1453-1460.  
10848958 B.A.van der Veen, J.C.Uitdehaag, B.W.Dijkstra, and L.Dijkhuizen (2000).
The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity.
  Eur J Biochem, 267, 3432-3441.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.