research papers
Crystal structures of Klebsiella pneumoniae pullulanase (KPP) in complex with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were refined at around 1.98–2.59 Å resolution from data collected at SPring-8. In the structures of the complexes obtained with 1 mM α-CD or γ-CD, one molecule of CD was found at carbohydrate-binding module 41 only (CBM41). In the structures of the complexes obtained with 1 mM β-CD or with 10 mM α-CD or γ-CD, two molecules of CD were found at CBM41 and in the active-site cleft, where the hydrophobic residue of Phe746 occupies the inside cavity of the CD rings. In contrast to α-CD and γ-CD, one β-CD molecule was found at the active site only in the presence of 0.1 mM β-CD. These results were coincident with the solution experiments, which showed that β-CD inhibits this enzyme more than a thousand times more potently than α-CD and γ-CD. The strong inhibition of β-CD is caused by the optimized interaction between β-CD and the side chain of Phe746. The increased Ki values of the F746A mutant for β-CD supported the importance of Phe746 in the strong interaction of pullulanase with β-CD.
Supporting information
Portable Document Format (PDF) file https://doi.org/10.1107/S2059798318014523/jc5015sup1.pdf |
PDB references: Klebsiella pneumoniae pullulanase, apo, space group P43212, 5yn2; complex formed in the presence of 0.1 mM β-cyclodextrin, 5yn7; complex formed in the presence of 1 mM α-cyclodextrin, 5yna; complex formed in the presence of 1 mM β-cyclodextrin, 5ync; complex formed in the presence of 1 mM γ-cyclodextrin, 5ynd; complex formed in the presence of 10 mM α-cyclodextrin, 5yne; complex formed in the presence of 10 mM γ-cyclodextrin, 5ynh