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J. Biol. Chem., Vol. 280, Issue 16, 16325-16334, April 22, 2005

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Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

CONTROLLED INCORPORATION OF THREE DISTINCT ENZYMES INTO A DEFINED TRIFUNCTIONAL SCAFFOLDIN^*

Henri-Pierre Fierobe, Florence Mingardon, Adva Mechaly¶, Anne Bélaïch, Marco T. Rincon||, Sandrine Pagès**, Raphael Lamed, Chantal Tardif**, Jean-Pierre Bélaïch**, and Edward A. Bayer¶

From the Bioénergétique et Ingénierie des Protéines, CNRS, Institut de Biologie Structurale et Microbiologie, Marseille 13402, France, the ^¶Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, the ^||Microbial Genetic Group, Rowett Research Institute, Aberdeen AB21 9SD, United Kingdom, the ^**Université de Provence, Marseille 13331, France, and the Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

Received for publication, December 22, 2004 , and in revised form, February 3, 2005.

^* This work was supported by the CNRS, the "Conseil Général des Bouches du Rhône," and the Region "Provence-Alpes-Côte d'Azur." Additional support was provided by Israel Science Foundation Grants 771/01 and 394/03 and by a grant from the United States-Israel Binational Science Foundation, Jerusalem, Israel. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Bioénergétique et Ingéniérie des Protéines, CNRS 31, Chemin Joseph Aiguier, 13402 Marseille, France. Tel.: 33-491-16-42-99; Fax: 33-491-71-33-21; E-mail: hpfierob{at}ibsm.cnrs-mrs.fr.

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