Recognition and hydrolysis of noncrystalline cellulose.
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.
Boraston
Alisdair B
AB
Protein Engineering Network of Centres of Excellence, PENCE Inc., National Business Centre, Edmonton, Alberta T6G 2S2, Canada. boraston@uvic.ca
Kwan
Emily
E
Chiu
Patrick
P
Warren
R Antony J
RA
Kilburn
Douglas G
DG
D000595:Amino Acid Sequence; D001407:Bacillus / Q000378:metabolism; D001667:Binding, Competitive; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002482:Cellulose; C407024:endoglucanase Cel5A; D002480:Cellulase