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

2018 ◽  
Vol 74 (11) ◽  
pp. 1115-1123 ◽  
Author(s):  
Naoki Saka ◽  
Hiroyuki Iwamoto ◽  
Dominggus Malle ◽  
Nobuyuki Takahashi ◽  
Kimihiko Mizutani ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Strong Interaction ◽  
Active Site ◽  
Side Chain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Strong Inhibition ◽  
Hydrophobic Residue ◽  
Active Site Cleft ◽  
Mechanism Of Interaction

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 K i values of the F746A mutant for β-CD supported the importance of Phe746 in the strong interaction of pullulanase with β-CD.

scholarly journals 1H, 13C and 15N backbone and side-chain assignment of a carbohydrate binding module from a xylanase from Roseburia intestinalis

2018 ◽  
Vol 13 (1) ◽  
pp. 55-58 ◽  
Author(s):  
Eva Madland ◽  
Yoshihito Kitaoku ◽  
Gerd Inger Sætrom ◽  
Maria Louise Leth ◽  
Morten Ejby ◽  
...  
Keyword(s):  
Side Chain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

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

2019 ◽  
Vol 75 (9) ◽  
pp. 792-803
Author(s):  
Naoki Saka ◽  
Dominggus Malle ◽  
Hiroyuki Iwamoto ◽  
Nobuyuki Takahashi ◽  
Kimihiko Mizutani ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Binding Affinity ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
Induced Fit ◽  
Structure And Activity ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706–710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706–710 and alters the binding affinity of the substrate.

scholarly journals The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

2006 ◽  
Vol 399 (3) ◽  
pp. 503-511 ◽  
Author(s):  
Akimasa Miyanaga ◽  
Takuya Koseki ◽  
Yozo Miwa ◽  
Yuichiro Mese ◽  
Sachiko Nakamura ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Side Chain ◽  
Titration Calorimetry ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Assays ◽  
The Family ◽  
Affinity Gel Electrophoresis ◽  
Hydrolysis Of

α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

scholarly journals Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

2009 ◽  
Vol 192 (2) ◽  
pp. 483-493 ◽  
Author(s):  
Shosuke Yoshida ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Unknown Function ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Carbohydrate Binding Module ◽  
Active Site Residues ◽  
Acetyl Xylan Esterase ◽  
Binding Studies

ABSTRACT Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.

Signature Active Site Architectures Illuminate the Molecular Basis for Ligand Specificity in Family 35 Carbohydrate Binding Module,

Biochemistry ◽  
2010 ◽  
Vol 49 (29) ◽  
pp. 6193-6205 ◽  
Author(s):  
Márcia A. S. Correia ◽  
D. Wade Abbott ◽  
Tracey M. Gloster ◽  
Vânia O. Fernandes ◽  
José A. M. Prates ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Ligand Specificity

Structure of the ectodomain of the electron transporter Rv2874 fromMycobacterium tuberculosisreveals a thioredoxin-like domain combined with a carbohydrate-binding module

2016 ◽  
Vol 72 (1) ◽  
pp. 40-48 ◽  
Author(s):  
David C. Goldstone ◽  
Peter Metcalf ◽  
Edward N. Baker
Keyword(s):  
Active Site ◽  
Family Members ◽  
Thioredoxin Reductase ◽  
Cellular Membrane ◽  
Integral Membrane Proteins ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Active Site Motif ◽  
Carbohydrate Binding Site ◽  
Reducing Potential

The members of the CcdA family are integral membrane proteins that use a disulfide cascade to transport electrons from the thioredoxin–thioredoxin reductase system in the interior of the cell into the extracytoplasmic space. The core transmembrane portion of this family is often elaborated with additional hydrophilic domains that act as adapters to deliver reducing potential to targets outside the cellular membrane. To investigate the function of family members inMycobacterium tuberculosis, the structure of the C-terminal ectodomain from Rv2874, one of three CcdA-family members present in the genome, was determined. The crystal structure, which was refined at 1.9 Å resolution withR= 0.195 andRfree= 0.219, reveals the predicted thioredoxin-like domain with its conserved Cys-X-X-Cys active-site motif. Unexpectedly, this domain is combined with a second domain with a carbohydrate-binding module (CBM) fold, this being the first reported example of a CBM in association with a thioredoxin-like domain fold. A cavity in the CBM adjacent to the thioredoxin active site suggests a likely carbohydrate-binding site, representing a broadening of the substrate range for CcdA-family members and an expansion of the thioredoxin-domain functionality to carbohydrate modification.

scholarly journals Carbohydrate-binding modules targeting branched polysaccharides: overcoming side-chain recalcitrance in a non-catalytic approach

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jiawen Liu ◽  
Di Sun ◽  
Jingrong Zhu ◽  
Cong Liu ◽  
Weijie Liu
Keyword(s):  
Side Chain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Enzymatic Conversion ◽  
Type Iii ◽  
Debranching Enzymes ◽  
Ligand Type ◽  
Carbohydrate Binding Modules ◽  
Hydrolysis Of ◽  
Main Strategy

AbstractExtensive decoration of backbones is a major factor resulting in resistance of enzymatic conversion in hemicellulose and other branched polysaccharides. Employing debranching enzymes is the main strategy to overcome this kind of recalcitrance at present. A carbohydrate-binding module (CBM) is a contiguous amino acid sequence that can promote the binding of enzymes to various carbohydrates, thereby facilitating enzymatic hydrolysis. According to previous studies, CBMs can be classified into four types based on their preference in ligand type, where Type III and IV CBMs prefer to branched polysaccharides than the linear and thus are able to specifically enhance the hydrolysis of substrates containing side chains. With a role in dominating the hydrolysis of branched substrates, Type III and IV CBMs could represent a non-catalytic approach in overcoming side-chain recalcitrance.

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