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J. Biol. Chem., Vol. 282, Issue 16, 12066-12074, April 20, 2007

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A Tomato Endo--1,4-glucanase, SlCel9C1, Represents a Distinct Subclass with a New Family of Carbohydrate Binding Modules (CBM49)^*

Breeanna R. Urbanowicz¹, Carmen Catalá, Diana Irwin, David B. Wilson, Daniel R. Ripoll^¶, and Jocelyn K. C. Rose²

From the Departments of Plant Biology and Molecular Biology and Genetics and the ^¶Computational Biology Service Unit at the Cornell Theory Center, Cornell University, Ithaca, New York 14853

A critical structural feature of many microbial endo--1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.

Received for publication, August 18, 2006 , and in revised form, January 26, 2007.

^* This work was supported in part by United States Department of Agriculture NRI Award 2002-35304-12680 (to J. K. C. R. and C. C.) and by grants from the United States National Science Foundation Plant Genome Program (DBI-0606595) and New York State Office of Science, Technology, and Academic Research (NYSTAR). This research was conducted in part with the resources of the Cornell Theory Center, which receives funding from Cornell University, New York State, Federal agencies, foundations, and corporate partners. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ Supported by United States Department of Agriculture Initiative for Future Agricultural and Food Systems Multidisciplinary Graduate Education Traineeship Fellowship 2001-52014-11484.

² To whom correspondence should be addressed: Dept. of Plant Biology, 228 Plant Science Bldg., Cornell University, Ithaca, NY 14853. Tel.: 607-255-4781; Fax: 607-255-5407; E-mail: jr286{at}cornell.edu.

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				B. R. Urbanowicz, A. B. Bennett, E. del Campillo, C. Catala, T. Hayashi, B. Henrissat, H. Hofte, S. J. McQueen-Mason, S. E. Patterson, O. Shoseyov, et al. Structural Organization and a Standardized Nomenclature for Plant Endo-1,4-beta-Glucanases (Cellulases) of Glycosyl Hydrolase Family 9 Plant Physiology, August 1, 2007; 144(4): 1693 - 1696. [Full Text] [PDF]