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  • Title: Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products.
    Author: Knegtel RM, Strokopytov B, Penninga D, Faber OG, Rozeboom HJ, Kalk KH, Dijkhuizen L, Dijkstra BW.
    Journal: J Biol Chem; 1995 Dec 08; 270(49):29256-64. PubMed ID: 7493956.
    Abstract:
    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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