Details

Autor(en) / Beteiligte

• Knegtel, R M
• Strokopytov, B
• Penninga, D
• Faber, O G
• Rozeboom, H J
• Kalk, K H
• Dijkhuizen, L
• Dijkstra, B W

Titel

Crystallographic Studies of the Interaction of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251 with Natural Substrates and Products

Ist Teil von

• The Journal of biological chemistry, 1995-12, Vol.270 (49), p.29256-29264

Ort / Verlag

United States: American Society for Biochemistry and Molecular Biology

Erscheinungsjahr

1995

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 α-cyclodextrin. Crystal structures were determined of wild type CGTase soaked at elevated pH with α-cyclodextrin (resolution, 2.1 Å) and maltoheptaose (2.4 Å). In addition, structures at cryogenic temperature were solved of the unliganded enzyme (2.2 Å) and of the D229N/E257Q mutant after soaking with α-cyclodextrin (2.6 Å). In the crystals soaked in α-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 α-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.