Elucidation of the mechanism of interaction between Klebsiella pneumoniae pullulanase and cyclodextrin

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 m M 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 m M β‐CD or with 10 m M 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 m M 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 K i values of the F746A mutant for β‐CD supported the importance of Phe746 in the strong interaction of pullulanase with β‐CD.