Essential role of a family‐32 carbohydrate‐binding module in substrate recognition by <i>Clostridium thermocellum</i> mannanase <i>Ct</i>Man5A

Mizutani, Kimiya; Sakka, Makiko; Kimura, Takeshi; Sakka, Kazuo

doi:10.1016/j.febslet.2014.03.022

Cited by 16 publications

(10 citation statements)

References 22 publications

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“…CBM32 was previously shown to directly participate in the substrate recognition required for catalytic action [23]; this was consistent with the current substrate specificity results for the TM1 recombinant protein. We also verified that both CBM9 and CBM32 could enhance the recognition of alginate lyase for polyG and increase the DP of the final degradation products; this may result from the substrate preference of CBM9 and CBM32, since different CBMs could recognize and bind to different carbohydrate.…”

Section: Discussionsupporting

confidence: 90%

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

et al. 2019

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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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Section: Discussionsupporting

confidence: 90%

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

et al. 2019

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“…Because CBM has the role of locating the crystalline face of cellulose, it is one of the rate‐limiting factors in the degradation process. Therefore, many physical,3–5 spectroscopic6–8 and computational modeling9–11 methods have been used to study the biological and structural properties of these proteins. To date, there are 71 known families of CBM proteins deposited in the CAZy database,12 classified according to amino‐acid sequence homology.…”

Section: Introductionmentioning

confidence: 99%

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Ivanir-Dabora

Nimerovsky

Madhu

et al. 2015

Chemistry A European J

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Magic-angle spinning solid-state NMR spectroscopy has been applied to study the dynamics of CBM3b-Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146-residue protein has a nine-stranded β-sandwich fold, in which 35 % of the residues are in the β-sheet and the remainder are composed of loops and turns. Dynamically averaged (1) H-(13) C dipolar coupling order parameters were extracted in a site-specific manner by using a pseudo-three-dimensional constant-time recoupled separated-local-field experiment (dipolar-chemical shift correlation experiment; DIPSHIFT). The backbone-Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water-mediated contacts, whereas other crystal contacts remain rigid.

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“…CBMs enhance the activity of their cognate enzymes by either disruption and modification of polysaccharide structures (24) or increasing enzyme concentration in the vicinity of substrate and sometimes by targeting specific carbohydrate structures in a native biomass containing different polysaccharides (2,25). Mizutani et al (26) showed that a CBM32 appended to a GH5 from Clostridium thermocellum directly influenced the mode of catalysis, resulting in difference in product formation in the presence and absence of CBM32. However, comparison of the hydrolyzed products generated by PeCsn and GH8 at a very early phase of degradation of 0% DA chitosan did not show any noticeable difference in the pattern and quantity of product formation (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Specificity of PeCBM32-Many ligand molecules like galactose, lactose, sialic acid, LacNAc, chitosan oligomer, and mannan oligomer are recognized by CBM32 appended to different enzymes (7,16,26). We studied the influence of DA of chitosan polymers on the binding of PeCBM32.…”

Section: Discussionmentioning

confidence: 99%

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii

Das¹,

Wagenknecht

Nareddy³

et al. 2016

Journal of Biological Chemistry

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We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.

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Essential role of a family‐32 carbohydrate‐binding module in substrate recognition by Clostridium thermocellum mannanase CtMan5A

Cited by 16 publications

References 22 publications

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

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

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii

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