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Title
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Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products.
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Authors
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R.M.Knegtel,
B.Strokopytov,
D.Penninga,
O.G.Faber,
H.J.Rozeboom,
K.H.Kalk,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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J Biol Chem, 1995,
270,
29256-29264.
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PubMed id
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Abstract
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Asp-229, Glu-257, and Asp-328 constitute the catalytic residues in cyclodextrin
glycosyl transferase from Bacillus circulans strain 251. Via site-directed
mutagenesis constructed D229N, E257Q, and D328N mutant proteins showed a
4,000-60,000-fold reduction of cyclization activity. A D229N/E257Q double mutant
showed a 700,000-fold reduction and was crystallized for use in soaking
experiments with alpha-cyclodextrin. Crystal structures were determined of wild
type CGTase soaked at elevated pH with alpha-cyclodextrin (resolution, 2.1 A)
and maltoheptaose (2.4 A). In addition, structures at cryogenic temperature were
solved of the unliganded enzyme (2.2 A) and of the D229N/E257Q mutant after
soaking with alpha-cyclodextrin (2.6 A). In the crystals soaked in
alpha-cyclodextrin and maltoheptaose, a maltotetraose molecule is observed to
bind in the active site. Residue 229 is at hydrogen bonding distance from the
C-6 hydroxyl group of the sugar, which after cleavage will contain the new
reducing end. In the D229N/E257Q double mutant structure, two
alpha-cyclodextrins are observed to replace two maltoses at the E-domain, thus
providing structural information on product inhibition via binding to the
enzyme's raw starch binding domain.
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