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PDBsum entry 1dtu
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Rational design of cyclodextrin glycosyltransferase from bacillus circulans strain 251 to increase alpha-Cyclodextrin production.
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Authors
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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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Ref.
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J Mol Biol, 2000,
296,
1027-1038.
[DOI no: ]
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PubMed id
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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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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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Secondary reference #1
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Title
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Engineering of cyclodextrin product specificity and ph optima of the thermostable cyclodextrin glycosyltransferase from thermoanaerobacterium thermosulfurigenes em1.
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Authors
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R.D.Wind,
J.C.Uitdehaag,
R.M.Buitelaar,
B.W.Dijkstra,
L.Dijkhuizen.
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Ref.
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J Biol Chem, 1998,
273,
5771-5779.
[DOI no: ]
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PubMed id
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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.
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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.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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Structure of cyclodextrin glycosyltransferase complexed with a maltononaose inhibitor at 2.6 angstrom resolution. Implications for product specificity.
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Authors
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B.Strokopytov,
R.M.Knegtel,
D.Penninga,
H.J.Rozeboom,
K.H.Kalk,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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Biochemistry, 1996,
35,
4241-4249.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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Site-Directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from bacillus circulans strain 251 affect activity and product specificity.
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Authors
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D.Penninga,
B.Strokopytov,
H.J.Rozeboom,
C.L.Lawson,
B.W.Dijkstra,
J.Bergsma,
L.Dijkhuizen.
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Ref.
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Biochemistry, 1995,
34,
3368-3376.
[DOI no: ]
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PubMed id
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