PDBsum entry 1cgv

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Glycosyltransferase PDB id
Protein chain
686 a.a. *
MAL ×3
_CA ×2
Waters ×171
* Residue conservation analysis
PDB id:
Name: Glycosyltransferase
Title: Site directed mutations of the active site residue tyrosine cyclodextrin glycosyltransferase from bacillus circulans st affecting activity and product specificity
Structure: Cyclomaltodextrin glucanotransferase. Chain: a. Engineered: yes. Mutation: yes
Source: Bacillus circulans. Organism_taxid: 1397
2.50Å     R-factor:   0.155    
Authors: B.V.Strokopytov,B.W.Dijkstra
Key ref:
D.Penninga et al. (1995). Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity. Biochemistry, 34, 3368-3376. PubMed id: 7880832 DOI: 10.1021/bi00010a028
05-Aug-94     Release date:   27-Feb-95    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P43379  (CDGT2_BACCI) -  Cyclomaltodextrin glucanotransferase
713 a.a.
686 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  


DOI no: 10.1021/bi00010a028 Biochemistry 34:3368-3376 (1995)
PubMed id: 7880832  
Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.
D.Penninga, B.Strokopytov, H.J.Rozeboom, C.L.Lawson, B.W.Dijkstra, J.Bergsma, L.Dijkhuizen.
Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)

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.  
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.  
18726621 R.M.Ong, K.M.Goh, N.M.Mahadi, O.Hassan, R.N.Rahman, and R.M.Illias (2008).
Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli.
  J Ind Microbiol Biotechnol, 35, 1705-1714.  
17891389 Z.Li, M.Wang, F.Wang, Z.Gu, G.Du, J.Wu, and J.Chen (2007).
gamma-Cyclodextrin: a review on enzymatic production and applications.
  Appl Microbiol Biotechnol, 77, 245-255.  
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
14993702 M.Akita, Y.Hatada, Y.Hidaka, Y.Ohta, M.Takada, Y.Nakagawa, K.Ogawa, T.Nakakuki, S.Ito, and K.Horikoshi (2004).
Crystallization and preliminary X-ray study of gamma-type cyclodextrin glycosyltransferase from Bacillus clarkii.
  Acta Crystallogr D Biol Crystallogr, 60, 586-587.  
12492486 H.Leemhuis, B.W.Dijkstra, and L.Dijkhuizen (2003).
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.
  Eur J Biochem, 270, 155-162.  
11397453 B.N.Gawande, and A.Y.Patkar (2001).
Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22.
  Enzyme Microb Technol, 28, 735-743.  
11828459 H.Driguez (2001).
Thiooligosaccharides as tools for structural biology.
  Chembiochem, 2, 311-318.  
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.  
11443082 M.Hemker, A.Stratmann, K.Goeke, W.Schröder, J.Lenz, W.Piepersberg, and H.Pape (2001).
Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp. strain SE50.
  J Bacteriol, 183, 4484-4492.  
11302176 T.Yokota, T.Tonozuka, S.Kamitori, and Y.Sakano (2001).
The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: effects of domain N on activity, specificity, stability and dimerization.
  Biosci Biotechnol Biochem, 65, 401-408.  
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.  
11064053 A.D.Blackwood, and C.Bucke (2000).
Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on cgtase.
  Enzyme Microb Technol, 27, 704-708.  
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.  
11150610 J.E.Nielsen, and T.V.Borchert (2000).
Protein engineering of bacterial alpha-amylases.
  Biochim Biophys Acta, 1543, 253-274.  
10574960 J.C.Uitdehaag, K.H.Kalk, B.A.van Der Veen, L.Dijkhuizen, and B.W.Dijkstra (1999).
The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution.
  J Biol Chem, 274, 34868-34876.
PDB code: 1d3c
9558324 A.K.Schmidt, S.Cottaz, H.Driguez, and G.E.Schulz (1998).
Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product beta-cyclodextrin.
  Biochemistry, 37, 5909-5915.
PDB code: 3cgt
10075634 M.Alcalde, F.J.Plou, E.Pastor, and A.Ballesteros (1998).
Effect of chemical modification of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. on its activity and product selectivity.
  Ann N Y Acad Sci, 864, 183-187.  
9488711 R.D.Wind, J.C.Uitdehaag, R.M.Buitelaar, B.W.Dijkstra, and L.Dijkhuizen (1998).
Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.
  J Biol Chem, 273, 5771-5779.
PDB code: 1a47
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.  
9648273 L.Bornaghi, J.P.Utille, Rekaï el-D, J.M.Mallet, P.Sinaÿ, and H.Driguez (1997).
Transfer reactions catalyzed by cyclodextrin glucosyltransferase using 4-thiomaltosyl and C-maltosyl fluorides as artificial donors.
  Carbohydr Res, 305, 561-568.  
8672460 B.Strokopytov, R.M.Knegtel, D.Penninga, H.J.Rozeboom, K.H.Kalk, L.Dijkhuizen, and B.W.Dijkstra (1996).
Structure of cyclodextrin glycosyltransferase complexed with a maltononaose inhibitor at 2.6 angstrom resolution. Implications for product specificity.
  Biochemistry, 35, 4241-4249.
PDB codes: 1dij 2dij
8955113 D.Penninga, B.A.van der Veen, R.M.Knegtel, S.A.van Hijum, H.J.Rozeboom, K.H.Kalk, B.W.Dijkstra, and L.Dijkhuizen (1996).
The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251.
  J Biol Chem, 271, 32777-32784.
PDB code: 1tcm
7493956 R.M.Knegtel, B.Strokopytov, D.Penninga, O.G.Faber, H.J.Rozeboom, K.H.Kalk, L.Dijkhuizen, and B.W.Dijkstra (1995).
Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products.
  J Biol Chem, 270, 29256-29264.
PDB codes: 1cxe 1cxf 1cxh 1cxi
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