scholarly journals Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

2007 ◽  
Vol 406 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Lavinia Cicortas Gunnarsson ◽  
Cedric Montanier ◽  
Richard B. Tunnicliffe ◽  
Mike P. Williamson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Hydrophobic Interactions ◽  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Rhodothermus Marinus ◽  
Binding Properties ◽  
Carbohydrate Binding Modules

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

scholarly journals The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

2001 ◽  
Vol 276 (51) ◽  
pp. 48580-48587 ◽  
Author(s):  
Mirjam Czjzek ◽  
David N. Bolam ◽  
Amor Mosbah ◽  
Julie Allouch ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Carbohydrate Binding ◽  
Ligand Binding Site ◽  
Carbohydrate Binding Modules ◽  
Common Sequence

Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase

2016 ◽  
Vol 473 (8) ◽  
pp. 1085-1095 ◽  
Author(s):  
Shoko Shinya ◽  
Shigenori Nishimura ◽  
Yoshihito Kitaoku ◽  
Tomoyuki Numata ◽  
Hisashi Kimoto ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Binding Site ◽  
Carbohydrate Binding ◽  
Binding Properties ◽  
Fungal Cell ◽  
Mutant Proteins ◽  
Loop Region ◽  
C Terminus ◽  
Carbohydrate Binding Modules ◽  
Paenibacillus Sp

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a β-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in 1H-15N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core β-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu14 of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg31, Tyr36 and Glu61 formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr36 of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr36 to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.

Advances in molecular engineering of carbohydrate-binding modules

2017 ◽  
Vol 85 (9) ◽  
pp. 1602-1617 ◽  
Author(s):  
Silvia Armenta ◽  
Silvia Moreno-Mendieta ◽  
Zaira Sánchez-Cuapio ◽  
Sergio Sánchez ◽  
Romina Rodríguez-Sanoja
Keyword(s):  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules

2019 ◽  
Vol 32 (9) ◽  
pp. 401-409
Author(s):  
Stefan Jarl Christensen ◽  
Silke Flindt Badino ◽  
Ana Mafalda Cavaleiro ◽  
Kim Borch ◽  
Peter Westh
Keyword(s):  
Substrate Surface ◽  
Soft Rot ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Chimeric Enzymes ◽  
Functional Differences ◽  
Steady State Kinetic ◽  
Carbohydrate Binding Modules ◽  
State Kinetic ◽  
Cellulose Surface

Abstract The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme’s ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

2001 ◽  
Vol 358 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Pichia Pastoris ◽  
Association Constant ◽  
Mutational Analysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Glycosylation Sites ◽  
High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

Clostridium thermocellumXyn10B Carbohydrate-Binding Module 22-2:  The Role of Conserved Amino Acids in Ligand Binding†,‡

Biochemistry ◽  
2001 ◽  
Vol 40 (31) ◽  
pp. 9167-9176 ◽  
Author(s):  
Hefang Xie ◽  
Harry J. Gilbert ◽  
Simon J. Charnock ◽  
Gideon J. Davies ◽  
Michael P. Williamson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Conserved Amino Acids

scholarly journals NMR Elucidation of Non-productive Binding Sites of 13C-Labeled Lignin Models with Carbohydrate Binding Module of Cellobiohydrolase I for Efficient Biomass Conversion

2020 ◽  
Author(s):  
Yuki Tokunaga ◽  
Takashi Nagata ◽  
Keiko Kondo ◽  
Masato Katahira ◽  
Takashi Watanabe
Keyword(s):  
Binding Sites ◽  
Hydrophobic Interactions ◽  
Biomass Conversion ◽  
Specific Interaction ◽  
Enzymatic Saccharification ◽  
Cost Effective ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Cellobiohydrolase I ◽  
Methoxy Groups

Abstract Background: Highly efficient enzymatic saccharification of pretreated lignocellulose is a primary key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase possessing carbohydrate binding module 1 (TrCBM1) as the C-terminal domain. Non-productive binding of TrCBM1 to lignin significantly decreases enzymatic saccharification efficiency and enhance cost of biomass conversion due to required additional enzymes. Understanding of the interaction mechanism between lignin and TrCBM1 is essentially required to realize cost-effective biofuels production, but the binding sites in lignin have not been clearly elucidated. Results: Three types of 13C-labeled b-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C HSQC spectra of the 13C-labeled lignin models exhibited that 13C-labels were correctly incorporated in the (1) aromatic rings and b positions, (2) a positions, and (3) methoxy groups, respectively. The TrCBM1 binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled b-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. This indicated that hydrophobic interactions and p–p stacking were dominating factors in non-productive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with C(II), which indicated that binding of TrCBM1 is evidently affected by configuration of lignin models. Long-chain lignins (DP 4.16–4.70) clearly bound to TrCBM1. Interactions with short-chain lignins (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion: The CSP analysis using 13C-labeled b-O-4 lignin oligomer models enabled us to identify TrCBM1 binding sites in lignin at the atomic level. This specific interaction analysis will lead to new molecular design of cellulase having controlled affinity to cellulose and lignin for cost-effective biorefinery process.

Homology-based model of the extracellular domain of the taste receptor T1R3

2002 ◽  
Vol 74 (7) ◽  
pp. 1117-1123 ◽  
Author(s):  
D. Eric Walters
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Metabotropic Glutamate Receptor ◽  
Hydrophobic Interactions ◽  
Ion Pairs ◽  
Taste Receptor ◽  
Docking Studies ◽  
Side Chains ◽  
Metabotropic Glutamate ◽  
Hydroxy Compounds

The extracellular ligand binding domain of the sweet receptor T1R3 has been homology-modeled on the basis of the crystal structure of the metabotropic glutamate receptor (mGluR1). The region of the model that corresponds to the ligand binding site of mGluR1 has numerous polar and charged side-chains, consistent with expectations for a site that would respond to poly hydroxy compounds such as mono- and disaccharides. Docking studies show that proposed active conformations of the high-potency sweeteners neotame, superaspartame, and SC-45647 could interact favorably in this binding site, forming ion pairs or ionic hydrogen bonds with His-163, Glu-318, and His-407, in addition to hydrophobic interactions with numerous nonpolar side-chains.

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