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 on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special "anchorage subunit." In this "anchor-enzyme" model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and that CipA contains nine repeated sequences with a cellulose binding 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), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing 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 crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.
Kruus
K
K
University of Rochester, Department of Chemical Engineering, NY 14627-0166, USA.
Lua
A C
AC
Demain
A L
AL
Wu
J H
JH
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D017931:DNA Primers; D004269:DNA, Bacterial; D008565:Membrane Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase