Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Biologia ◽  
2014 ◽  
Vol 69 (6) ◽  
Author(s):  
Darrell Cockburn ◽  
Casper Wilkens ◽  
Christian Ruzanski ◽  
Susan Andersen ◽  
Jonas Willum Nielsen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Binding Studies ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Functional Roles ◽  
Carbohydrate Binding Modules ◽  
The Many ◽  
Key Residues

AbstractSurface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

scholarly journals Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

2000 ◽  
Vol 345 (1) ◽  
pp. 53 ◽  
Author(s):  
Maher ABOU HACHEM ◽  
Eva NORDBERG KARLSSON ◽  
Eva BARTONEK-ROXÅ ◽  
Srinivasrao RAGHOTHAMA ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Carbohydrate Binding ◽  
Rhodothermus Marinus ◽  
Binding Studies ◽  
Carbohydrate Binding Modules

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Vincent Valk ◽  
Rachel M. van der Kaaij ◽  
Lubbert Dijkhuizen
Keyword(s):  
Protein Interactions ◽  
Catalytic Domain ◽  
Substrate Surface ◽  
Carbohydrate Binding ◽  
Protein Protein Interactions ◽  
Functional Roles ◽  
Cazy Database ◽  
Carbohydrate Binding Modules ◽  
Fibronectin Type Iii

AbstractFibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.

scholarly journals A bivalent glycopeptide to target two putative carbohydrate binding sites on FimH

2010 ◽  
Vol 6 ◽  
pp. 801-809 ◽  
Author(s):  
Thisbe K Lindhorst ◽  
Kathrin Bruegge ◽  
Andreas Fuchs ◽  
Oliver Sperling
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Carbohydrate Binding ◽  
Carbohydrate Recognition Domain ◽  
Carbohydrate Recognition ◽  
Binding Studies ◽  
Bivalent Ligand ◽  
X Ray ◽  
Carbohydrate Binding Site

FimH is a mannose-specific bacterial lectin found on type 1 fimbriae with a monovalent carbohydrate recognition domain (CRD) that is known from X-ray studies. However, binding studies with multivalent ligands have suggested an additional carbohydrate-binding site on this protein. In order to prove this hypothesis, a bivalent glycopeptide ligand with the capacity to bridge two putative carbohydrate binding sites on FimH was designed and synthesized. Anti-adhesion assays with the new bivalent ligand and type 1-fimbriated bacteria have revealed, that verification of the number of carbohydrate binding sites on FimH with a tailor-made bivalent glycopeptide requires further investigation to be conclusive.

scholarly journals Comparative Analysis of Carbohydrate Active Enzymes in the Flammulina velutipes var. lupinicola Genome

2020 ◽  
Vol 9 (1) ◽  
pp. 20
Author(s):  
Hye-Won Yu ◽  
Ji-Hoon Im ◽  
Won-Sik Kong ◽  
Young-Jin Park
Keyword(s):  
De Novo ◽  
Animal Feed ◽  
Gc Content ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Flammulina Velutipes ◽  
Carbohydrate Binding ◽  
Carbohydrate Active Enzymes ◽  
Carbohydrate Binding Modules ◽  
Genes Encoding

The purpose of this study was to determine the genome sequence of Flammulina velutipes var. lupinicola based on next-generation sequencing (NGS) and to identify the genes encoding carbohydrate-active enzymes (CAZymes) in the genome. The optimal assembly (71 kmer) based on ABySS de novo assembly revealed a total length of 33,223,357 bp (49.53% GC content). A total of 15,337 gene structures were identified in the F.velutipes var. lupinicola genome using ab initio gene prediction method with Funannotate pipeline. Analysis of the orthologs revealed that 11,966 (96.6%) out of the 15,337 predicted genes belonged to the orthogroups and 170 genes were specific for F. velutipes var. lupinicola. CAZymes are divided into six classes: auxiliary activities (AAs), glycosyltransferases (GTs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycoside hydrolases (GHs), and carbohydrate-binding modules (CBMs). A total of 551 genes encoding CAZymes were identified in the F. velutipes var. lupinicola genome by analyzing the dbCAN meta server database (HMMER, Hotpep, and DIAMOND searches), which consisted of 54–95 AAs, 145–188 GHs, 55–73 GTs, 6–19 PLs, 13–59 CEs, and 7–67 CBMs. CAZymes can be widely used to produce bio-based products (food, paper, textiles, animal feed, and biofuels). Therefore, information about the CAZyme repertoire of the F. velutipes var. lupinicola genome will help in understanding the lignocellulosic machinery and in-depth studies will provide opportunities for using this fungus for biotechnological and industrial applications.

scholarly journals Improved Characterization of the Solution Kinetics and Thermodynamics of Biotin, Biocytin and HABA Binding to Avidin and Streptavidin

2018 ◽  
Author(s):  
Roberto F. Delgadillo ◽  
Timothy C. Mueser ◽  
Kathia Zaleta-Rivera ◽  
Katie A. Carnes ◽  
José González-Valdez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Thermodynamic Cycle ◽  
Reaction Model ◽  
Strong Temperature Dependence ◽  
Surface Binding ◽  
Binding Studies ◽  
Diffusion Limited ◽  
Single Transition ◽  
Association Data

ABSTRACTThe high affinity (KD∼ 10−15M) of biotin to avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109M−1s−1) but recent single molecule and surface binding studies indicate they are slower than expected (105to 107M−1s−1). In this study, we asked whether these reactions in solution are diffusion controlled, what reaction model and thermodynamic cycle described the complex formation, and the functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)-benzoic acid. Native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The konvalues of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the konvalues revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). The data suggest that the avidin binding sites are deeper and less accessible than those of streptavidin. Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.

scholarly journals Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

2020 ◽  
pp. AEM.02634-20
Author(s):  
Mohamed A. Attia ◽  
Harry Brumer
Keyword(s):  
Binding Protein ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Carbohydrate Active Enzymes ◽  
Plant Polysaccharides ◽  
New Family ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11)

2013 ◽  
Vol 451 (2) ◽  
pp. 289-300 ◽  
Author(s):  
Aldino Viegas ◽  
João Sardinha ◽  
Filipe Freire ◽  
Daniel F. Duarte ◽  
Ana L. Carvalho ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Solution Structure ◽  
Close Contact ◽  
Van Der Waals Interactions ◽  
Backbone Dynamics ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Binding Studies ◽  
Carbohydrate Binding Modules ◽  
Wide Range

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11–cellohexaose and CtCBM11–cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand–protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

Hemophilic factor VIII C1- and C2-domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 979-987 ◽  
Author(s):  
Miao-Liang Liu ◽  
Betty W. Shen ◽  
Shelley Nakaya ◽  
Kathleen P. Pratt ◽  
Kazuo Fujikawa ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Homology Model ◽  
C2 Domain ◽  
Missense Mutations ◽  
Surface Binding ◽  
Binding Studies ◽  
Equivalent Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII C domains contain key binding sites for von Willebrand factor (vWF) and phospholipid membranes. Hemophilic patients were screened for factor VIII C-domain mutations to provide a well-characterized series. Mutated residues were localized to the high-resolution C2 structure and to a homology model of C1. Of 30 families found with mutations in the C domains, there were 14 missense changes, and 9 of these were novel. Of the missense mutations, 10 were associated with reduced vWF binding and 8 were at residues with surface-exposed side chains. Six of the 10 mutants had nearly equivalent factor VIII clotting activity and antigen level, suggesting that reduced vWF binding could cause hemophilia by reducing factor VIII stability in circulation. When the present series was combined with previously described mutations from an online international database, 11 C1 and C2 mutations in patients with mild or moderately severe hemophilia A were associated with antibody-inhibitor development in at least one affected individual. Of these substitutions, 6 occurred at surface-exposed residues. As further details of the C1 structure and its interface with C2 become available, and as binding studies are performed on the plasma of more patients with hemophilic C-domain mutations, prediction of surface binding sites should improve, allowing confirmation by site-specific mutagenesis of surface-exposed residues.

scholarly journals Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

1999 ◽  
Vol 345 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Maher ABOU HACHEM ◽  
Eva NORDBERG KARLSSON ◽  
Eva BARTONEK-ROXÅ ◽  
Srinivasrao RAGHOTHAMA ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Binding Affinities ◽  
Binding Assays ◽  
Rhodothermus Marinus ◽  
Binding Studies ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Cellulose Binding

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichiacoli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

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