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J. Biol. Chem., Vol. 278, Issue 8, 6120-6127, February 21, 2003
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From Cellulase Cel5A from alkalophilic
Bacillus sp. 1139 contains a family 17 carbohydrate-binding
module (BspCBM17) and a family 28 CBM
(BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for
binding are not conserved. BspCBM28 was obtained as a
discrete polypeptide by engineering the cel5A gene.
BspCBM17 could not be obtained as a discrete polypeptide,
so a family 17 CBM from endoglucanase Cel5A of Clostridium
cellulovorans, CcCBM17, was used to compare the
binding characteristics of the two families of CBM. Both
CcCBM17 and BspCBM28 recognized two classes of
binding sites on amorphous cellulose: a high affinity site
(Ka ~1 × 106
M
Recognition and Hydrolysis of Noncrystalline Cellulose*
§¶,§
,, and§
The Protein Engineering Network of Centres of
Excellence, PENCE Inc., National Business Centre, Edmonton, Alberta T6G
2S2, Canada, the Department of Microbiology and Immunology,
University of British Columbia, Vancouver, British Columbia V6T 1Z3,
Canada, and the § Biotechnology Laboratory, University of
British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
1) and a low affinity site
(Ka ~2 × 104
M1). They did not compete for binding to the
high affinity sites, suggesting that they bound at different sites on
the cellulose. A polypeptide, BspCBM17/CBM28, comprising
the tandem CBMs from Cel5A, bound to amorphous cellulose with a
significantly higher affinity than the sum of the affinities of
CcCBM17 and BspCBM28, indicating cooperativity
between the linked CBMs. Cel5A mutants were constructed that were
defective in one or both of the CBMs. The mutants differed from the
wild-type enzyme in the amounts and sizes of the soluble products
produced from amorphous cellulose. This suggests that either the CBMs
can modify the action of the catalytic module of Cel5A or that they
target the enzyme to areas of the cellulose that differ in
susceptibility to hydrolysis.
*
This work was supported by the Natural Sciences and
Engineering Research Council and the Protein Engineering Network
Centres of Excellence Research Council of Canada (to D. G. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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