The interaction of carbohydrate-binding modules with insoluble non-crystalline cellulose is enthalpically driven

Natural cellulose exists as a composite of cellulose forms, which can be broadly characterized as crystalline or non-crystalline. The recognition of both of these forms of cellulose by the CBMs (carbohydrate-binding modules) of microbial glycoside hydrolases is important for the efficient natural and biotechnological conversion of cellulosic biomass. The category of CBM that binds insoluble non-crystalline cellulose does so with an affinity approx. 10–20-fold greater than their affinity for cello-oligosaccharides and/or soluble polysaccharides. This phenomenon has been assumed to originate from the effects of changes in configurational entropy upon binding. The loss of configurational entropy is thought to be less profound upon binding to conformationally restrained insoluble non-crystalline cellulose, resulting in larger free energies of binding. However, using isothermal titration calorimetry, it is shown that this is not the case for the high-affinity interactions of CcCBM17 (the family 17 CBM from EngF of Clostridium cellulovorans) and BspCBM28 (the family 28 CBM from Cel5A of Bacillus species 1139) with regenerated cellulose, an insoluble preparation of primarily non-crystalline cellulose. The enhanced free energy of binding of non-crystalline cellulose relative to cello-oligosaccharides is by virtue of improved enthalpy, not entropy.

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The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

Biochemical Journal ◽

10.1042/bj20021860 ◽

2003 ◽

Vol 371 (3) ◽

pp. 1027-1043 ◽

Cited By ~ 82

Author(s):

Deborah HOGG ◽

Gavin PELL ◽

Paul DUPREE ◽

Florence GOUBET ◽

Susana M. MARTÍN-ORÚE ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Glycoside Hydrolases ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

Molecular Architecture ◽

Catalytic Domains ◽

Carbohydrate Binding Modules ◽

Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

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CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose

Journal of Bacteriology ◽

10.1128/jb.185.2.391-398.2003 ◽

2003 ◽

Vol 185 (2) ◽

pp. 391-398 ◽

Cited By ~ 93

Author(s):

Rachel Gilad ◽

Larisa Rabinovich ◽

Sima Yaron ◽

Edward A. Bayer ◽

Raphael Lamed ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Catalytic Domain ◽

Terminal Segment ◽

Glycoside Hydrolases ◽

General Nature ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

Truncated Form ◽

Cellulose Substrates ◽

The Family

ABSTRACT The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

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Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module

Biochemical Journal ◽

10.1042/bj3550155 ◽

2001 ◽

Vol 355 (1) ◽

pp. 155-165 ◽

Cited By ~ 16

Author(s):

Ian E. BROWN ◽

Marie H. MALLEN ◽

Simon J. CHARNOCK ◽

Gideon J. DAVIES ◽

Gary W. BLACK

Keyword(s):

Sequence Similarity ◽

Functional Module ◽

Pectate Lyase ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

High Sequence Identity ◽

Pectate Lyases ◽

Carbohydrate Binding Modules ◽

The Family ◽

Catalytic Module

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH10 and in the presence of 2mM Ca2+. Highest enzyme activity was detected at 62°C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167–177], are the first CBM-containing pectinases described to date.

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Structure of a family 3b′ carbohydrate-binding module from the Cel9V glycoside hydrolase fromClostridium thermocellum: structural diversity and implications for carbohydrate binding

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909043030 ◽

2009 ◽

Vol 66 (1) ◽

pp. 33-43 ◽

Cited By ~ 12

Author(s):

Svetlana Petkun ◽

Sadanari Jindou ◽

Linda J. W. Shimon ◽

Sonia Rosenheck ◽

Edward A. Bayer ◽

...

Keyword(s):

Structural Diversity ◽

Glycoside Hydrolases ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

Asymmetric Unit ◽

Space Group ◽

Carbohydrate Binding Modules ◽

Planar Array ◽

Binding Residues ◽

Cellulose Binding

Family 3 carbohydrate-binding modules (CBM3s) are associated with both cellulosomal scaffoldins and family 9 glycoside hydrolases (GH9s), which are multi-modular enzymes that act on cellulosic substrates. CBM3s bind cellulose. X-ray crystal structures of these modules have established an accepted cellulose-binding mechanism based on stacking interactions between the sugar rings of cellulose and a planar array of aromatic residues located on the CBM3 surface. These planar-strip residues are generally highly conserved, although some CBM3 sequences lack one or more of these residues. In particular, CBM3b′ fromClostridium thermocellumCel9V exhibits such sequence changes and fails to bind cellulosic substrates. A crystallographic investigation of CBM3b′ has been initiated in order to understand the structural reason(s) for this inability. CBM3b′ crystallized in space groupC2221(diffraction was obtained to 2.0 Å resolution in-house) with three independent molecules in the asymmetric unit and in space groupP41212 (diffraction was obtained to 1.79 Å resolution in-house and to 1.30 Å resolution at a synchrotron) with one molecule in the asymmetric unit. The molecular structure of Cel9V CBM3b′ revealed that in addition to the loss of several cellulose-binding residues in the planar strip, changes in the backbone create a surface `hump' which could interfere with the formation of cellulose–protein surface interactions and thus prevent binding to crystalline cellulose.

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A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011977 ◽

2020 ◽

Vol 295 (31) ◽

pp. 10638-10652

Author(s):

Ping Chen ◽

Ran Liu ◽

Mengmeng Huang ◽

Jinlu Zhu ◽

Dong Wei ◽

...

Keyword(s):

Wide Spectrum ◽

Streptococcus Suis ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Unique Combination ◽

Enzymatic Assays ◽

Tracheal Epithelial Cells ◽

Carbohydrate Binding Modules ◽

Host Signaling ◽

Important Host

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

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Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules

Microorganisms ◽

10.3390/microorganisms8040481 ◽

2020 ◽

Vol 8 (4) ◽

pp. 481 ◽

Cited By ~ 3

Author(s):

Toshihiko Katoh ◽

Miriam N. Ojima ◽

Mikiyasu Sakanaka ◽

Hisashi Ashida ◽

Aina Gotoh ◽

...

Keyword(s):

Carbon Sources ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Altruistic Behavior ◽

Carbohydrate Binding ◽

Human Gut ◽

Genetic Characteristics ◽

Beneficial Effects ◽

Carbohydrate Binding Modules ◽

Enzymatic Machinery

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.

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Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14015520 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1232-1235 ◽

Cited By ~ 2

Author(s):

Bruna Medeia Campos ◽

Thabata Maria Alvarez ◽

Marcelo Vizona Liberato ◽

Igor Polikarpov ◽

Harry J. Gilbert ◽

...

Keyword(s):

Fossil Fuels ◽

Plant Biomass ◽

Metagenomic Library ◽

Glycoside Hydrolases ◽

Diffusion Method ◽

Carbohydrate Binding ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Carbohydrate Binding Modules

In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space groupI23, with unit-cell parametersa=b=c= 88.07 Å.

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Essential role of the family-22 carbohydrate-binding modules for β-1,3-1,4-glucanase activity of Clostridium stercorarium Xyn10B

FEBS Letters ◽

10.1016/s0014-5793(04)00160-7 ◽

2004 ◽

Vol 561 (1-3) ◽

pp. 155-158 ◽

Cited By ~ 24

Author(s):

Rie Araki ◽

Mursheda K Ali ◽

Makiko Sakka ◽

Tetsuya Kimura ◽

Kazuo Sakka ◽

...

Keyword(s):

Essential Role ◽

Carbohydrate Binding ◽

Glucanase Activity ◽

Carbohydrate Binding Modules ◽

The Family

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Genome Sequencing and Carbohydrate-Active Enzyme (CAZyme) Repertoire of the White Rot Fungus Flammulina elastica

International Journal of Molecular Sciences ◽

10.3390/ijms19082379 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2379 ◽

Cited By ~ 12

Author(s):

Young-Jin Park ◽

Yong-Un Jeong ◽

Won-Sik Kong

Keyword(s):

Gene Prediction ◽

Gc Content ◽

Industrial Applications ◽

Fungal Species ◽

Amino Acid Sequences ◽

Glycoside Hydrolases ◽

White Rot ◽

White Rot Fungus ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

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