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J Bacteriol, April 1998, p. 1709-1714, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis

Diana Irwin, Dong-Hoon Shin,dagger Sheng Zhang, Brian K. Barr,Dagger Joshua Sakon,§ P. Andrew Karplus, and David B. Wilson*

Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Received 3 November 1997/Accepted 27 January 1998

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

* Corresponding author. Mailing address: BMCB, Biotechnology Building, Cornell University, Ithaca, NY 14853. Phone: (607) 255-5708. Fax: (607) 255-2428. E-mail: dbw3{at}cornell.edu.

dagger Present address: Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, Jochiwon, Choong Nam 339-700, Korea.

Dagger Present address: Department of Chemistry, Loyola College, Baltimore, MD 21210.

§ Present address: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701.




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