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PDBsum entry 1dtu
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* Residue conservation analysis
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Enzyme class:
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E.C.2.4.1.19
- cyclomaltodextrin glucanotransferase.
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Reaction:
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Degrades starch to cyclodextrins by formation of a 1,4-alpha-D- glucosidic bond.
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DOI no:
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J Mol Biol
296:1027-1038
(2000)
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PubMed id:
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Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.
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B.A.van der Veen,
J.C.Uitdehaag,
D.Penninga,
G.J.van Alebeek,
L.M.Smith,
B.W.Dijkstra,
L.Dijkhuizen.
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ABSTRACT
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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.
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Selected figure(s)
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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
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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.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2000,
296,
1027-1038)
copyright 2000.
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Figures were
selected
by an automated process.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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H.Leemhuis,
R.M.Kelly,
and
L.Dijkhuizen
(2010).
Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications.
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Appl Microbiol Biotechnol,
85,
823-835.
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P.Fernandes
(2010).
Enzymes in food processing: a condensed overview on strategies for better biocatalysts.
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Enzyme Res,
2010,
862537.
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R.M.Kelly,
L.Dijkhuizen,
and
H.Leemhuis
(2009).
The evolution of cyclodextrin glucanotransferase product specificity.
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Appl Microbiol Biotechnol,
84,
119-133.
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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.
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Appl Microbiol Biotechnol,
83,
483-490.
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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.
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Appl Biochem Biotechnol,
146,
3.
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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.
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J Biol Chem,
283,
10727-10734.
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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.
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Appl Environ Microbiol,
71,
5823-5827.
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Q.Qi,
and
W.Zimmermann
(2005).
Cyclodextrin glucanotransferase: from gene to applications.
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Appl Microbiol Biotechnol,
66,
475-485.
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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.
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Proteins,
54,
128-134.
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PDB code:
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H.Leemhuis,
B.W.Dijkstra,
and
L.Dijkhuizen
(2003).
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.
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Eur J Biochem,
270,
155-162.
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T.P.Frandsen,
M.M.Palcic,
and
B.Svensson
(2002).
Substrate recognition by three family 13 yeast alpha-glucosidases.
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Eur J Biochem,
269,
728-734.
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E.A.MacGregor,
S.Janecek,
and
B.Svensson
(2001).
Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.
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Biochim Biophys Acta,
1546,
1.
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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.
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Proteins,
43,
327-335.
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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.
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Appl Environ Microbiol,
67,
1453-1460.
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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.
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Eur J Biochem,
267,
3432-3441.
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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.
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