Physical association of the catalytic and helper modules of a family‐9 glycoside hydrolase is essential for activity

Burstein, Tal; Shulman, Michal; Jindou, Sadanari; Petkun, Svetlana; Frolow, Felix; Shoham, Yuval; Bayer, Edward A.; Lamed, Raphael

doi:10.1016/j.febslet.2009.02.013

Cited by 47 publications

(49 citation statements)

References 23 publications

(32 reference statements)

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“…These domains are structurally linked to the catalytic domain and may be considered as part of it. As a consequence, deletion of the ancillary contiguous domain usually generates an inactive form of the GH9 enzyme (44), whereas in other families of glycoside hydrolases the catalytic domain is a module that remains generally active even when it is produced individually. The recurrent physical association of the GH9 catalytic domains and their neighboring ancillary domains may thus have been selected to generate a larger array of activity patterns, further diversified by addition of supplementary modules/domains such as the CBM3b or CBM4, which are not specific of GH9 enzymes as they are found in bacterial scaffoldins and glycoside hydrolases classified in other GH families.…”

Section: Discussionmentioning

confidence: 99%

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Ravachol

Borne

Tardif

et al. 2014

Journal of Biological Chemistry

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Background: Family-9 glycoside hydrolases displaying various organizations are plethoric in cellulosome-producing bacteria. Results: The 13 family-9 enzymes synthesized by Clostridium cellulolyticum exhibit different substrate specificities, activities, and relationships with key cellulosomal cellulases in artificial cellulosomes. Conclusion: This variety is required for efficient degradation of crystalline cellulose. Significance: This first global picture provides insights on the multiplicity and diversity of family-9 enzymes in bacterial cellulosomes.

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Section: Discussionmentioning

confidence: 99%

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Ravachol

Borne

Tardif

et al. 2014

Journal of Biological Chemistry

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“…This alternative mode of interaction with cellulose is reflected in the sequences of CBM3c, which diverge from those of CBM3a and CBM3b. CBM3c is fused tightly to the catalytic module by numerous intermodular contacts to ensure proper orientation of the two modules, so they can be considered a single functional unit (12,23,26,31,44).…”

Section: Cloning and Expression Of Recombinant Proteinsmentioning

confidence: 99%

Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States

Vazana¹,

Moraïs²,

Barak

et al. 2010

Appl Environ Microbiol

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The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

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“…CBMs have since been shown to be critical for the processivity of processive GH9 EGs (8,(13)(14)(15). For example, crystalline cellulose degradation and processivity were abolished by deletion of CBM3c of Thermomonospora fusca (16), the main function of which is to disrupt the cellulose chains in the crystalline cellulose substrate and to feed a single chain into the active site of the CD (8).…”

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confidence: 99%

Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

Zheng

Ding

2013

Appl Environ Microbiol

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EG1 is a modular glycoside hydrolase family 5 endoglucanase from Volvariella volvacea consisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.

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Physical association of the catalytic and helper modules of a family‐9 glycoside hydrolase is essential for activity

Cited by 47 publications

References 23 publications

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States

Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

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