Recognition and Hydrolysis of Noncrystalline Cellulose
| Autor: | P. Chiu, Emily Kwan, R. Antony J. Warren, Alisdair B. Boraston, Douglas G. Kilburn |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Protein Conformation Molecular Sequence Data Bacillus Cooperativity Cellulase Binding Competitive Biochemistry chemistry.chemical_compound Hydrolysis Escherichia coli Amino Acid Sequence Cloning Molecular Binding site Cellulose Molecular Biology Clostridium cellulovorans chemistry.chemical_classification Sequence Homology Amino Acid biology Cell Biology Hydrogen-Ion Concentration biology.organism_classification Amino acid Kinetics Enzyme chemistry biology.protein Crystallization Sequence Alignment Plasmids |
| Zdroj: | Journal of Biological Chemistry. 278:6120-6127 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m209554200 |
| Popis: | 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 (K(a) approximately 1 x 10(6) M(-1)) and a low affinity site (K(a) approximately 2 x 10(4) M(-1)). 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. |
| Databáze: | OpenAIRE |
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