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PDBsum entry 1cgx
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Glycosyltransferase
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PDB id
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1cgx
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References listed in PDB file
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Key reference
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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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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
78%.
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Abstract
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Tyrosine 195 is located in the center of the active site cleft of cyclodextrin
glycosyltransferase (EC 2.4.1.19) 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)
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Secondary reference #1
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Title
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Nucleotide sequence and X-Ray structure of cyclodextrin glycosyltransferase from bacillus circulans strain 251 in a maltose-Dependent crystal form.
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Authors
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C.L.Lawson,
R.Van montfort,
B.Strokopytov,
H.J.Rozeboom,
K.H.Kalk,
G.E.De vries,
D.Penninga,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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J Mol Biol, 1994,
236,
590-600.
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PubMed id
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Secondary reference #2
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Title
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Maltodextrin-Dependent crystallization of cyclomaltodextrin glucanotransferase from bacillus circulans.
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Authors
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C.L.Lawson,
J.Bergsma,
P.M.Bruinenberg,
G.De vries,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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J Mol Biol, 1990,
214,
807-809.
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PubMed id
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