Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

ABSTRACTXyn30D from the xylanolytic strainPaenibacillus barcinonensishas been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing aKmof 14.72 mg/ml and akcatvalue of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

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A mannanase, ManA, of the polycentric anaerobic fungusOrpinomycessp. strain PC-2 has carbohydrate binding and docking modules

Canadian Journal of Microbiology ◽

10.1139/w05-033 ◽

2005 ◽

Vol 51 (7) ◽

pp. 559-568 ◽

Cited By ~ 17

Author(s):

Eduardo A Ximenes ◽

Huizhong Chen ◽

Irina A Kataeva ◽

Michael A Cotta ◽

Carlos R Felix ◽

...

Keyword(s):

Specific Activity ◽

High Specific Activity ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Amino Acid Residues ◽

Anaerobic Fungus ◽

Ph Profile ◽

Catalytic Module ◽

Cellulose Binding

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or β-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 °C, but was rapidly inactivated at 60 °C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.Key words: Orpinomyces, anaerobic fungi, mannanase, cellulose-binding module, cellulosome.

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Structure of the catalytic module and the family 13 carbohydrate binding module of a family 10 xylanase from Strepromyces olivaceoviridis in complex with xylose and galactose

Carbohydrate Bioengineering - Special Publications ◽

10.1039/9781847550323-00106 ◽

2010 ◽

pp. 106-112

Author(s):

Z. Fujimoto ◽

A. Kuno ◽

S. Kaneko ◽

H. Kobayashi ◽

I. Kusakabe ◽

...

Keyword(s):

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

The Family ◽

Catalytic Module ◽

Family 10 Xylanase

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Effect of Family 22 Carbohydrate-Binding Module on the Thermostability of Xyn10B Catalytic Module fromClostridium stercorarium

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.60348 ◽

2006 ◽

Vol 70 (12) ◽

pp. 3039-3041 ◽

Cited By ~ 14

Author(s):

Rie ARAKI ◽

Shuichi KARITA ◽

Akiyoshi TANAKA ◽

Tetsuya KIMURA ◽

Kazuo SAKKA

Keyword(s):

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Catalytic Module

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DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

Applied and Environmental Microbiology ◽

10.1128/aem.00446-16 ◽

2016 ◽

Vol 82 (16) ◽

pp. 4975-4981 ◽

Cited By ~ 22

Author(s):

Lorena Rodríguez-Rubio ◽

Hans Gerstmans ◽

Simon Thorpe ◽

Stéphane Mesnage ◽

Rob Lavigne ◽

...

Keyword(s):

Enzymatic Activity ◽

Thermal Resistance ◽

High Performance ◽

Antimicrobial Agents ◽

Biochemical Characterization ◽

Specific Activity ◽

Three Dimensional ◽

Dimensional Structure ◽

Gram Negative ◽

Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

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Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.02009-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7048-7059 ◽

Cited By ~ 26

Author(s):

Libin Ye ◽

Xiaoyun Su ◽

George E. Schmitz ◽

Young Hwan Moon ◽

Jing Zhang ◽

...

Keyword(s):

Specific Activity ◽

Cellulose Hydrolysis ◽

Thermophilic Bacterium ◽

Carbohydrate Binding ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

C Terminus ◽

Carbohydrate Binding Modules ◽

Β Sheets ◽

Biochemical Analyses

ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.

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Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass

Scientific Reports ◽

10.1038/s41598-017-03927-y ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 10

Author(s):

Nandita Pasari ◽

Nidhi Adlakha ◽

Mayank Gupta ◽

Zeenat Bashir ◽

Girish H. Rajacharya ◽

...

Keyword(s):

Catalytic Activity ◽

Plant Biomass ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module

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

Marine Drugs ◽

10.3390/md18010025 ◽

2019 ◽

Vol 18 (1) ◽

pp. 25 ◽

Cited By ~ 4

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.

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Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

Journal of Bacteriology ◽

10.1128/jb.00503-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5424-5436 ◽

Cited By ~ 13

Author(s):

Shosuke Yoshida ◽

Charles W. Hespen ◽

Robert L. Beverly ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Plant Cell Wall ◽

Catalytic Efficiency ◽

Glycoside Hydrolases ◽

Site Directed Mutagenesis ◽

Biofuel Production ◽

Carbohydrate Binding ◽

Fibrobacter Succinogenes ◽

Carbohydrate Binding Module ◽

Degrading Bacterium ◽

Catalytic Module

ABSTRACT Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k cat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

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

Applied and Environmental Microbiology ◽

10.1128/aem.07457-11 ◽

2012 ◽

Vol 78 (14) ◽

pp. 4781-4787 ◽

Cited By ~ 23

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.

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