A tomato endo-beta-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49).
A critical structural feature of many microbial endo-beta-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.
Urbanowicz
Breeanna R
BR
Department of Plant Biology, Cornell Theory Center, Cornell University, Ithaca, New York 14853, USA.
Catalá
Carmen
C
Irwin
Diana
D
Wilson
David B
DB
Ripoll
Daniel R
DR
Rose
Jocelyn K C
JK
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry*; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002482:Cellulose / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D002482:Cellulose; D005982:Glutathione Transferase; D002480:Cellulase; C090774:endoglucanase EGL1