Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 11, Pages 7335-7348Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.545046
Keywords
Carbohydrate; Cellulase; Enzyme Kinetics; Enzymes; Protein Complexes; GH9; Cellulose; Cellulosome; Clostridium cellulolyticum; Synergy
Categories
Funding
- Ministere de l'Enseignement Superieur et de la Recherche
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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. The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.
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