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Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens

Marco T. Rincon,¹ Shi-You Ding,^2,3, Sheila I. McCrae,¹ Jennifer C. Martin,¹ Vincenzo Aurilia,⁴ Raphael Lamed,³ Yuval Shoham,^5, Edward A. Bayer,² and Harry J. Flint^1*

Gut Microbiology Group, Rowett Research Institute, Aberdeen, United Kingdom,¹ Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,² Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv,³ Department of Food Engineering and Biotechnology,⁵ Institute of Catalysis Science and Technology, Technion—Israel Institute of Technology, Haifa, Israel,⁶ Institute of Protein Biochemistry, National Research Council, Naples, Italy⁴

Received 5 July 2002/ Accepted 30 September 2002

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

For a commentary on this article, see page 701 in this issue.

Present address: National Renewable Energy Laboratory, Biotechnology Center for Fuels and Chemicals, Golden, CO 80401.

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