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

Abstract

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.

Relationship between the induced-fit loop and the activity of Klebsiella pneumoniae pullulanase

“…The gene for the rKPP-G680L mutant was constructed by a double-PCR method. The PCR reaction was performed in a reaction volume totaling 50 ml and containing 5 U PfuTurbo Hotstart DNA polymerase (Agilent Technologies), 10Â cloned Pfu reaction buffer, 500 mM dNTP, 400 nmol ml À1 of two synthesized mutagenesis primers and 25 ng ml À1 pCold-TF vector (Takara Bio); the pCold-TF vector included the rKPP gene, which had previously been constructed (Saka et al, 2018). The above-described primers used the forward primer 5 0 -TTCGACCTGATGCTGTACCACCCGAAAGC-3 0 and the reverse primer 5 0 -TGCGCTTTCGGGTGGTACAGCAT CAGGTC-3 0 .…”

“…The expression systems for rKPP and the G680L mutant have been reported previously (Saka et al, 2018). The crude solution was applied onto a cobalt ion immobilized chelating Sepharose Fast Flow column (GE Healthcare Life Sciences) equilibrated with 500 mM sodium chloride containing 50 mM Tris-HCl buffer pH 8.0.…”

“…Based on structure-based sequence alignment, there are three deletions (parts of L2, L6b and L8) and one insertion (between 1b and 1) in GH13_14 compared with GH13_13 (Stam et al, 2006;Mikami et al, 2006). To date, we have reported structures of pullulanase from Klebsiella pneumoniae (hereafter referred to as KPP) in a ligand-free form and in complexes with saccharides (G1, G2, G3, G4 and isoG2) and with -,and -cyclodextrins (Mikami et al, 2006;Saka et al, 2018). KPP is a commercial enzyme produced by Hayashibara Co. which was derived from K. pneumoniae strain ATCC 9621 and consists of five domains (CBM41, N2, CBM48, A and C).…”

“…The sequence of region II of KPP is GFRFDLMLY; however, the sequence in other GH13_13 enzymes is GFRFDLMGY(H). Recently, we cloned recombinant pullulanase (hereafter referred to as rKPP), which has a glycine in region II, as in other GH13_13 enzymes, from another strain of K. pneumoniae, ATCC 9621 (Saka et al, 2018).…”

“…KPP is reported to perform an induced-fit motion of the active-site loop (residues 706-710) included in the conserved region III upon the binding of maltooligosaccharides (Mikami et al, 2006;Saka et al, 2018). In contrast to KPP, the loop conformation of limit dextrinase from Hordeum vulgare (HvLD;Vester-Christensen et al, 2010;Møller et al, 2015) was reported to be unchanged between the ligand-free structure and the complexed structure.…”