PDBsum entry 1a47

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Glycosidase PDB id
Protein chain
683 a.a. *
_CA ×2
Waters ×185
* Residue conservation analysis
PDB id:
Name: Glycosidase
Title: Cgtase from thermoanaerobacterium thermosulfurigenes em1 in complex with a maltohexaose inhibitor
Structure: Cyclodextrin glycosyltransferase. Chain: a. Synonym: cgtase. Engineered: yes
Source: Thermoanaerobacterium thermosulfurigenes. Organism_taxid: 33950. Strain: em1. Cellular_location: extracellular. Gene: amya. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.56Å     R-factor:   0.191     R-free:   0.250
Authors: J.C.M.Uitdehaag,K.H.Kalk,H.J.Rozeboom,B.W.Dijkstra
Key ref:
R.D.Wind et al. (1998). Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J Biol Chem, 273, 5771-5779. PubMed id: 9488711 DOI: 10.1074/jbc.273.10.5771
11-Feb-98     Release date:   17-Jun-98    
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

 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  


DOI no: 10.1074/jbc.273.10.5771 J Biol Chem 273:5771-5779 (1998)
PubMed id: 9488711  
Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.
R.D.Wind, J.C.Uitdehaag, R.M.Buitelaar, B.W.Dijkstra, L.Dijkhuizen.
The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.
  Selected figure(s)  
Figure 2.
Fig. 2. Conformation of the maltohexaose inhibitor in the active site of the CGTase from T. thermosulfurigenes EM1. The inhibitor is occupying subsites 3 to +3 in domains A and B of the CGTase.
Figure 3.
Fig. 3. Superposition of the maltohexaose (sticks) and maltononaose (lines) inhibitor structures. At subsite +3 the conformation of the maltohexaose inhibitor is more bent toward Phe^196 and is stabilized by Lys47, which is Arg47 in the CGTase from B. circulans strain 251. Moreover, the replacement of Tyr89 (B. circulans CGTase) by Asp89 (T. thermosulfurigenes EM1 CGTase) makes that the "straight" maltononaose conformation at subsite +3 is not as stably bound^ in T. thermosulfurigenes EM1 CGTase than as in B. circulans CGTase.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 5771-5779) copyright 1998.  
  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.  
19682075 C.Christiansen, M.Abou Hachem, S.Janecek, A.Viksø-Nielsen, A.Blennow, and B.Svensson (2009).
The carbohydrate-binding module family 20--diversity, structure, and function.
  FEBS J, 276, 5006-5029.  
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.  
18626642 Y.H.Liu, F.P.Lu, Y.Li, J.L.Wang, and C.Gao (2008).
Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations.
  Appl Microbiol Biotechnol, 80, 795-803.  
15939021 L.W.Yang, and I.Bahar (2005).
Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes.
  Structure, 13, 893-904.  
12492486 H.Leemhuis, B.W.Dijkstra, and L.Dijkhuizen (2003).
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.
  Eur J Biochem, 270, 155-162.  
12554949 H.W.Choe, K.S.Park, J.Labahn, J.Granzin, C.J.Kim, and G.Büldt (2003).
Crystallization and preliminary X-ray diffraction studies of alpha-cyclodextrin glucanotransferase isolated from Bacillus macerans.
  Acta Crystallogr D Biol Crystallogr, 59, 348-349.  
11916711 A.Tomschy, R.Brugger, M.Lehmann, A.Svendsen, K.Vogel, D.Kostrewa, S.F.Lassen, D.Burger, A.Kronenberger, A.P.van Loon, L.Pasamontes, and M.Wyss (2002).
Engineering of phytase for improved activity at low pH.
  Appl Environ Microbiol, 68, 1907-1913.  
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.  
10651801 B.A.van der Veen, G.J.van Alebeek, J.C.Uitdehaag, B.W.Dijkstra, and L.Dijkhuizen (2000).
The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.
  Eur J Biochem, 267, 658-665.  
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.  
  11082203 I.Przylas, Y.Terada, K.Fujii, T.Takaha, W.Saenger, and N.Sträter (2000).
X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus. Implications for the synthesis of large cyclic glucans.
  Eur J Biochem, 267, 6903-6913.
PDB code: 1esw
10869182 J.C.Uitdehaag, G.J.van Alebeek, B.A.van Der Veen, L.Dijkhuizen, and B.W.Dijkstra (2000).
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.
  Biochemistry, 39, 7772-7780.
PDB codes: 1eo5 1eo7
11150610 J.E.Nielsen, and T.V.Borchert (2000).
Protein engineering of bacterial alpha-amylases.
  Biochim Biophys Acta, 1543, 253-274.  
10679895 N.Ishii, K.Haga, K.Yamane, and K.Harata (2000).
Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution.
  J Mol Recognit, 13, 35-43.
PDB code: 1d7f
9860832 R.Mosi, H.Sham, J.C.Uitdehaag, R.Ruiterkamp, B.W.Dijkstra, and S.G.Withers (1998).
Reassessment of acarbose as a transition state analogue inhibitor of cyclodextrin glycosyltransferase.
  Biochemistry, 37, 17192-17198.  
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.