The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome

K. Kruus, A. C. Lua, A. L. Demain, J. H.D. Wu*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

56 Citations (Scopus)

Abstract

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 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 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.

Original languageEnglish
Pages (from-to)9254-9258
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number20
DOIs
Publication statusPublished - 12 Oct 1995
MoE publication typeA1 Journal article-refereed

Keywords

  • cellulase
  • cellulose degradation
  • CelS
  • protein complex

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