THE ANCHORAGE FUNCTION OF CIPA (CELL), A SCAFFOLDING PROTEIN OF THE CLOSTRIDIUM-THERMOCELLUM CELLULOSOME

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate, Based o n our studies of the cellulase complex of Clostridium thermocellum (th e cellulosome), we have previously proposed that such binding can be b rought about by a special ''anchorage subunit,'' In this ''anchor-enzy me'' model, CipA (a major subunit of the cellulosome) enhances the act ivity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface, We have subsequently report ed that CelS contains a conserved duplicated sequence at its C terminu s and that CipA contains nine repeated sequences with a cellulose bind ing domain (CBD) in between the second and third repeats, In this work , we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), an d CBD/R3, expressed in Escherichia coli, As analyzed by nondenaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD, Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD /R3, Such anchorage apparently increased the rCelS activity toward cry stalline cellulose, These results substantiate the proposed anchor-enz yme model and the expected roles of individual CipA domains and the co nserved duplicated sequence of CelS.