Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum.
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.
From the Aix-Marseille Université-CNRS, Laboratoire de Chimie Bactérienne UMR7283, Institut de Microbiologie de la Méditerranée, Marseille Cedex 20, France.
Julie
J
Ravachol
Romain
R
Borne
Chantal
C
Tardif
Pascale
P
de Philip
Henri-Pierre
HP
Fierobe
D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D044603:Cellulosomes / Q000737:chemistry*; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology*; D003912:Dextrins / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D016680:Genome, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010802:Phylogeny; D011485:Protein Binding; D015202:Protein Engineering; D013379:Substrate Specificity; D014783:Viscosity
D003912:Dextrins; D002482:Cellulose; C068994:cellodextrin; D006026:Glycoside Hydrolases; D002480:Cellulase
Carbohydrate; Cellulase; Cellulose; Cellulosome; Clostridium cellulolyticum; Enzyme Kinetics; Enzymes; GH9; Protein Complexes; Synergy