scholarly journals Structural insights into the mechanisms and specificities of IgG-active endoglycosidases

Glycobiology ◽  
2019 ◽  
Vol 30 (4) ◽  
pp. 268-279 ◽  
Author(s):  
Jonathan J Du ◽  
Erik H Klontz ◽  
Marcelo E Guerin ◽  
Beatriz Trastoy ◽  
Eric J Sundberg
Keyword(s):  
Bond Cleavage ◽  
Point Mutations ◽  
Antibody Engineering ◽  
Chemoenzymatic Synthesis ◽  
Structural Basis ◽  
Carbohydrate Binding ◽  
Effector Functions ◽  
Carbohydrate Binding Modules ◽  
Structural Insights ◽  
Made In

Abstract The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.

scholarly journals The Structural Basis for the Ligand Specificity of Family 2 Carbohydrate-binding Modules

2000 ◽  
Vol 275 (52) ◽  
pp. 41137-41142 ◽  
Author(s):  
Peter J. Simpson ◽  
Hefang Xie ◽  
David N. Bolam ◽  
Harry J. Gilbert ◽  
Michael P. Williamson
Keyword(s):  
Structural Basis ◽  
Carbohydrate Binding ◽  
Ligand Specificity ◽  
Carbohydrate Binding Modules

scholarly journals A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin

2020 ◽  
Vol 295 (27) ◽  
pp. 9134-9146 ◽  
Author(s):  
Sophanit Mekasha ◽  
Tina Rise Tuveng ◽  
Fatemeh Askarian ◽  
Swati Choudhary ◽  
Claudia Schmidt-Dannert ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Bond Cleavage ◽  
Hydrolytic Enzymes ◽  
Hydrolytic Activity ◽  
Full Length ◽  
Spatial Proximity ◽  
Carbohydrate Binding ◽  
Lytic Polysaccharide Monooxygenase ◽  
Chitin Binding Domain ◽  
Carbohydrate Binding Modules

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and β-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens. Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants—JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18—indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

scholarly journals Structural basis for carbohydrate-binding specificity—A comparative assessment of two engineered carbohydrate-binding modules

Glycobiology ◽  
2012 ◽  
Vol 22 (7) ◽  
pp. 948-961 ◽  
Author(s):  
Laura von Schantz ◽  
Maria Håkansson ◽  
Derek T Logan ◽  
Björn Walse ◽  
Jacob Österlin ◽  
...  
Keyword(s):  
Binding Specificity ◽  
Comparative Assessment ◽  
Structural Basis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components

2013 ◽  
Vol 288 (23) ◽  
pp. 16827-16838 ◽  
Author(s):  
Orly Salama-Alber ◽  
Maroor K. Jobby ◽  
Seth Chitayat ◽  
Steven P. Smith ◽  
Bryan A. White ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Calcium Binding ◽  
Plant Cell Wall ◽  
Structural Basis ◽  
Single Type ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Surface Attachment ◽  
Carbohydrate Binding Modules

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

scholarly journals Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

2021 ◽  
pp. 100638
Author(s):  
Marie Sofie Møller ◽  
Souad El Bouaballati ◽  
Bernard Henrissat ◽  
Birte Svensson
Keyword(s):  
Functional Diversity ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3175
Author(s):  
Mariana Barbosa ◽  
Hélvio Simões ◽  
Duarte Miguel F. Prazeres
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Protein A ◽  
Chemical Properties ◽  
Main Idea ◽  
Bioactive Molecules ◽  
Scaffolding Protein ◽  
Carbohydrate Binding ◽  
Biological Interactions ◽  
Carbohydrate Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

scholarly journals Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hong-Hsing Liu ◽  
Yu-Chen Lin ◽  
Chen-Shuan Chung ◽  
Kevin Liu ◽  
Ya-Hui Chang ◽  
...  
Keyword(s):  
Markov Models ◽  
Protein Domains ◽  
Degradation Pathway ◽  
The Other ◽  
Carbohydrate Binding ◽  
Gram Stain ◽  
Active Protein ◽  
Metabolic Potential ◽  
Independent Validation ◽  
Carbohydrate Binding Modules

AbstractBowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10−4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10−5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

2017 ◽  
Vol 474 (20) ◽  
pp. 3373-3389 ◽  
Author(s):  
Dong-Dong Meng ◽  
Xi Liu ◽  
Sheng Dong ◽  
Ye-Fei Wang ◽  
Xiao-Qing Ma ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Complex Structure ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Substrate Selectivity ◽  
Glucose Residue ◽  
Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

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