title: Effect of linker length and dockerin position on conversion of a Thermobifida fusca endoglucanase to the cellulosomal mode.
abstract: We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant "cellulosomized" enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Caspi
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.forename: Rachel
authors.initials: R
authors.lastname: Haimovitz
authors.forename: Diana
authors.initials: D
authors.lastname: Irwin
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: David B
authors.initials: DB
authors.lastname: Wilson
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D002482:Cellulose; C407024:endoglucanase Cel5A; D002480:Cellulase