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PDBsum entry 1tcm

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protein metals Protein-protein interface(s) links
Glycosyltransferase PDB id
1tcm
Jmol
Contents
Protein chains
686 a.a. *
Metals
_CA ×4
Waters ×265
* Residue conservation analysis
PDB id:
1tcm
Name: Glycosyltransferase
Title: Cyclodextrin glycosyltransferase w616a mutant from bacillus circulans strain 251
Structure: Cyclodextrin glycosyltransferase. Chain: a, b. Synonym: cgtase. Engineered: yes. Mutation: yes
Source: Bacillus circulans. Organism_taxid: 1397. Strain: 251. Expressed in: bacillus circulans. Expression_system_taxid: 1397.
Resolution:
2.20Å     R-factor:   0.193     R-free:   0.250
Authors: R.M.A.Knegtel,B.W.Dijkstra
Key ref:
D.Penninga et al. (1996). The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J Biol Chem, 271, 32777-32784. PubMed id: 8955113 DOI: 10.1074/jbc.271.51.32777
Date:
07-Oct-96     Release date:   21-Apr-97    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
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 1 residue position (black cross)

 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: 10.1074/jbc.271.51.32777 J Biol Chem 271:32777-32784 (1996)
PubMed id: 8955113  
 
 
The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251.
D.Penninga, B.A.van der Veen, R.M.Knegtel, S.A.van Hijum, H.J.Rozeboom, K.H.Kalk, B.W.Dijkstra, L.Dijkhuizen.
 
  ABSTRACT  
 
The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules. Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain. To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site. beta-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain. CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 A resolution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes. The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.
 
  Selected figure(s)  
 
Figure 2.
Fig. 2. Construction of plasmid pDP66K. Subcloning steps are indicated adjacent to the arrows.
Figure 8.
Fig. 8. Electron density in 2|F[o] F[c]| maps contoured at 1 for residues A614-A618 and B614-B618, labeled A and^ B, respectively. Residues adjacent to the mutated tryptophan have been indicated. The replacement of Trp-616 by Ala is confirmed^ by the electron density in this region.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1996, 271, 32777-32784) copyright 1996.  
  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.  
20159465 N.M.Koropatkin, and T.J.Smith (2010).
SusG: a unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules.
  Structure, 18, 200-215.
PDB codes: 3k8k 3k8l 3k8m
19968859 N.Z.Wayllace, H.A.Valdez, R.A.Ugalde, M.V.Busi, and D.F.Gomez-Casati (2010).
The starch-binding capacity of the noncatalytic SBD2 region and the interaction between the N- and C-terminal domains are involved in the modulation of the activity of starch synthase III from Arabidopsis thaliana.
  FEBS J, 277, 428-440.  
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.  
19333997 J.Øbro, I.Sørensen, P.Derkx, C.T.Madsen, M.Drews, M.Willer, J.D.Mikkelsen, and W.G.Willats (2009).
High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.
  Proteomics, 9, 1861-1868.  
19139240 M.Palomo, S.Kralj, M.J.van der Maarel, and L.Dijkhuizen (2009).
The unique branching patterns of Deinococcus glycogen branching enzymes are determined by their N-terminal domains.
  Appl Environ Microbiol, 75, 1355-1362.  
19367403 R.M.Kelly, L.Dijkhuizen, and H.Leemhuis (2009).
The evolution of cyclodextrin glucanotransferase product specificity.
  Appl Microbiol Biotechnol, 84, 119-133.  
19052787 R.Rodríguez-Sanoja, N.Oviedo, L.Escalante, B.Ruiz, and S.Sánchez (2009).
A single residue mutation abolishes attachment of the CBM26 starch-binding domain from Lactobacillus amylovorus alpha-amylase.
  J Ind Microbiol Biotechnol, 36, 341-346.  
18611383 N.M.Koropatkin, E.C.Martens, J.I.Gordon, and T.J.Smith (2008).
Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices.
  Structure, 16, 1105-1115.
PDB codes: 3ck7 3ck8 3ck9 3ckb 3ckc
18704190 S.Jemli, E.Ben Messaoud, S.Ben Mabrouk, and S.Bejar (2008).
The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme.
  J Biomed Biotechnol, 2008, 692573.  
17803687 S.Bozonnet, M.T.Jensen, M.M.Nielsen, N.Aghajari, M.H.Jensen, B.Kramhøft, M.Willemoës, S.Tranier, R.Haser, and B.Svensson (2007).
The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity.
  FEBS J, 274, 5055-5067.
PDB codes: 2qps 2qpu
16862594 N.Palopoli, M.V.Busi, M.S.Fornasari, D.Gomez-Casati, R.Ugalde, and G.Parisi (2006).
Starch-synthase III family encodes a tandem of three starch-binding domains.
  Proteins, 65, 27-31.  
17068753 N.Wang, Y.Zhang, Q.Wang, J.Liu, H.Wang, Y.Xue, and Y.Ma (2006).
Gene cloning and characterization of a novel alpha-amylase from alkaliphilic Alkalimonas amylolytica.
  Biotechnol J, 1, 1258-1265.  
16262690 M.Machovic, B.Svensson, E.A.MacGregor, and S.Janecek (2005).
A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21.
  FEBS J, 272, 5497-5513.  
15630515 Q.Qi, and W.Zimmermann (2005).
Cyclodextrin glucanotransferase: from gene to applications.
  Appl Microbiol Biotechnol, 66, 475-485.  
15640201 R.Rodríguez-Sanoja, B.Ruiz, J.P.Guyot, and S.Sanchez (2005).
Starch-binding domain affects catalysis in two Lactobacillus alpha-amylases.
  Appl Environ Microbiol, 71, 297-302.  
15939348 R.Rodríguez-Sanoja, N.Oviedo, and S.Sánchez (2005).
Microbial starch-binding domain.
  Curr Opin Microbiol, 8, 260-267.  
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
12562794 G.van Keulen, A.N.Ridder, L.Dijkhuizen, and W.G.Meijer (2003).
Analysis of DNA binding and transcriptional activation by the LysR-type transcriptional regulator CbbR of Xanthobacter flavus.
  J Bacteriol, 185, 1245-1252.  
12492486 H.Leemhuis, B.W.Dijkstra, and L.Dijkhuizen (2003).
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.
  Eur J Biochem, 270, 155-162.  
12581203 S.Janecek, B.Svensson, and E.A.MacGregor (2003).
Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.
  Eur J Biochem, 270, 635-645.  
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.  
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.  
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.  
10913296 J.Kormos, P.E.Johnson, E.Brun, P.Tomme, L.P.McIntosh, C.A.Haynes, and D.G.Kilburn (2000).
Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis.
  Biochemistry, 39, 8844-8852.  
10793202 L.M.Hamilton, C.T.Kelly, and W.M.Fogarty (2000).
Review: cyclodextrins and their interaction with amylolytic enzymes.
  Enzyme Microb Technol, 26, 561-567.  
11093706 Y.K.Kim, and J.F.Robyt (2000).
Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules.
  Carbohydr Res, 328, 509-515.  
  10508102 K.Ohdan, T.Kuriki, H.Kaneko, J.Shimada, T.Takada, Z.Fujimoto, H.Mizuno, and S.Okada (1999).
Characteristics of two forms of alpha-amylases and structural implication.
  Appl Environ Microbiol, 65, 4652-4658.  
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.  
9365988 P.M.Coutinho, and P.J.Reilly (1997).
Glucoamylase structural, functional, and evolutionary relationships.
  Proteins, 29, 334-347.  
9245426 R.Mosi, S.He, J.Uitdehaag, B.W.Dijkstra, and S.G.Withers (1997).
Trapping and characterization of the reaction intermediate in cyclodextrin glycosyltransferase by use of activated substrates and a mutant enzyme.
  Biochemistry, 36, 9927-9934.  
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 codes are shown on the right.