scholarly journals Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: Structural and functional significance of the intermodular linker

2015 ◽  
Author(s):  
Svetlana Petkun ◽  
Inna Rozman Grinberg ◽  
Raphael Lamed ◽  
Sadanari Jindou ◽  
Tal Burstein ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Spatial Organization ◽  
Cellulase Activity ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Endoglucanase Activity ◽  
Carbohydrate Binding Modules ◽  
Processive Endoglucanase ◽  
Catalytic Module

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

scholarly journals Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: Structural and functional significance of the intermodular linker

2015 ◽  
Author(s):  
Svetlana Petkun ◽  
Inna Rozman Grinberg ◽  
Raphael Lamed ◽  
Sadanari Jindou ◽  
Tal Burstein ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Spatial Organization ◽  
Cellulase Activity ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Endoglucanase Activity ◽  
Carbohydrate Binding Modules ◽  
Processive Endoglucanase ◽  
Catalytic Module

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

scholarly journals Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

2012 ◽  
Vol 78 (14) ◽  
pp. 4781-4787 ◽  
Author(s):  
Kimiya Mizutani ◽  
Vânia O. Fernandes ◽  
Shuichi Karita ◽  
Ana S. Luís ◽  
Makiko Sakka ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Guar Gum ◽  
Catalytic Domain ◽  
Hypothetical Protein ◽  
Carbohydrate Binding ◽  
Type I ◽  
Content Type ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Product Profile

ABSTRACTIn general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis.Clostridium thermocellumhypothetical protein Cthe_0821, defined here asC. thermocellumMan5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes whenC. thermocellumis cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity.C. thermocellumMan5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 ofC. thermocellumMan5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity ofC. thermocellumMan5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile ofC. thermocellumMan5A is affected by the presence of CBM32.

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

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.

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers

2004 ◽  
Vol 326 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Lesley McCartney ◽  
Harry J Gilbert ◽  
David N Bolam ◽  
Alisdair B Boraston ◽  
J.Paul Knox
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Molecular Probes ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cell Wall Polymers

scholarly journals Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

2015 ◽  
Vol 81 (6) ◽  
pp. 2006-2014 ◽  
Author(s):  
Dong-Dong Meng ◽  
Yu Ying ◽  
Xiao-Hua Chen ◽  
Ming Lu ◽  
Kang Ning ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Glycoside Hydrolase ◽  
Residual Activity ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Evolutionary Strategy ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Carbohydrate Binding Modules ◽  
Interdomain Interactions

ABSTRACTXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified fromCaldicellulosiruptorsp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed inEscherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol−1versus 94.7 U nmol−1) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.

scholarly journals Metagenomic analysis reveals rumen microbiota alteration of the yak at different stages of growth

2021 ◽  
Author(s):  
Li Wang ◽  
Li Juan ◽  
Ahmad Aboragah ◽  
Mingfeng Jiang ◽  
Juan J. Loor
Keyword(s):  
Glycoside Hydrolase ◽  
Age Groups ◽  
Carbohydrate Binding ◽  
The Tibetan Plateau ◽  
Tissue Slices ◽  
Rumen Microbiota ◽  
Stages Of Growth ◽  
Ruminant Species ◽  
Cazy Database ◽  
Carbohydrate Binding Modules

Abstract The Yak (Bos grunniens) is a unique ruminant species that is crucially important to agriculture in the Tibetan plateau. Variation of microorganism communities in the yak rumen is of great interest because of possible links to environmentally and economically important traits. In this study, we performed histological and microbial analyses of the yak rumen at 5 stages of growth: 1 day, 20 days, 60 days, 15 months, and 5 years of age. Tissue slices and metagenomics sequencing were used. The rumen index increased gradually from 1 day to 5 years of age. These were significant differences in rumen index between the 60d, 15m, and 5y group (p < 0.05). Compared with other time points, the thickness of muscularis along with length and width of rumen papillae at 60 d,15 m, and 5 years of age increased and differed (p < 0.05), respectively. At the phylum level, Bacteroidetes and Firmicutes were the phyla with the highest abundance in all the age groups. A total of 115,401 genes were annotated on the CAZy database. Glycoside Hydrolase (GH) had the highest relative abundance, followed by Glycosyl Transferase (GT), and Carbohydrate-binding Modules (CBM). There were significant variations for the microbial species and CAZys within the five groups. Taken together, the morphology and microbiota in the yak rumen changed at various stages of growth and likely played a significant role in the absorption of nutrients. This study provides new insights into the function of yak rumen microbiota and physiologic adaptations in plateau animals.

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.

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.

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