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

2001 ◽  
Vol 355 (1) ◽  
pp. 155-165 ◽  
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.

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

2005 ◽  
Vol 385 (2) ◽  
pp. 479-484 ◽  
Author(s):  
Alisdair B. BORASTON
Keyword(s):  
Configurational Entropy ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Titration Calorimetry ◽  
Bacillus Species ◽  
Crystalline Cellulose ◽  
Regenerated Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
The Family

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.

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

FEBS Letters ◽  
2004 ◽  
Vol 561 (1-3) ◽  
pp. 155-158 ◽  
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

Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan

2007 ◽  
Vol 8 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Lada Filonova ◽  
Åsa M. Kallas ◽  
Lionel Greffe ◽  
Gunnar Johansson ◽  
Tuula T. Teeri ◽  
...  
Keyword(s):  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Pulp Fibers ◽  
Carbohydrate Binding Modules

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

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
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.

scholarly journals Function of the Family-9 and Family-22 Carbohydrate-Binding Modules in a Modular β-1,3-1,4-Glucanase/Xylanase Derived fromClostridium stercorariumXyn10B

2005 ◽  
Vol 69 (8) ◽  
pp. 1562-1567 ◽  
Author(s):  
Guangshan ZHAO ◽  
Ehsan ALI ◽  
Rie ARAKI ◽  
Makiko SAKKA ◽  
Tetsuya KIMURA ◽  
...  
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
The Family

Two structurally related starch-binding domain families CBM25 and CBM26

Biologia ◽  
2014 ◽  
Vol 69 (9) ◽  
Author(s):  
Katarína Majzlová ◽  
Štefan Janeček
Keyword(s):  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Bacillus Halodurans ◽  
Carbohydrate Binding ◽  
Structural Comparison ◽  
Raw Starch ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Cazy Database ◽  
The Family

AbstractStarch binding domains (SBDs) are able to bind to and facilitate the degradation of raw starch and starchy substrates. In general, in the CAZy database they have been classified among the carbohydrate-binding module (CBM) families. The two families CBM25 and CBM26 together with families CBM20, 21, 34, 41, 45, 48, 53, 58, 68 and 69 belong to twelve SBD CAZy families. They represent a group of closely related modules exhibiting some sequence similarity, although each of the two families possesses its own features. Both CBM25 and CBM26 adopt a typical SBD fold of distorted β-barrel as recognized in the modules present in the maltohexaose-producing amylase from Bacillus halodurans. With regard to catalytic domains, most members are α-amylases and maltooligosaccharide-producing amylases from the α-amylase glycoside hydrolase (GH) family GH13, but also some β-amylases (GH14) and hypothetical proteins (e.g. from the family GH31) are known. The main goal of this review was to compare the available amino acid sequences of SBDs from both families CBM25 and CBM26 and to reveal, if possible, SBD(s) with the character “intermediary” between the CBM25 and CBM26. Emphasis was also given on a structural comparison of the identified intermediary SBD with the CBM25 and CBM26 representatives and a detailed evolutionary division of both CBM families that can be utilized for defining the future subfamilies.

scholarly journals Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 25 ◽  
Author(s):  
Zhelun Zhang ◽  
Luyao Tang ◽  
Mengmeng Bao ◽  
Zhigang Liu ◽  
Wengong Yu ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Functional Characterization ◽  
Alginate Lyase ◽  
Carbohydrate Binding ◽  
Enzyme Degradation ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

scholarly journals Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules

2019 ◽  
Vol 116 (45) ◽  
pp. 22545-22551 ◽  
Author(s):  
Vera Novy ◽  
Kevin Aïssa ◽  
Fredrik Nielsen ◽  
Suzana K. Straus ◽  
Peter Ciesielski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Degradation Mechanism ◽  
Length Distribution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Distribution Analysis ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

scholarly journals Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jordan F. Russell ◽  
Matthew L. Russo ◽  
Xuewen Wang ◽  
Neal Hengge ◽  
Daehwan Chung ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Protein Glycosylation ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Unique Gene ◽  
Glycosyl Transferase ◽  
Caldicellulosiruptor Bescii ◽  
Carbohydrate Binding Modules

ABSTRACT Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. IMPORTANCE Caldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

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