Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10cellulose-binding module from Pseudomonas xylanase A to insoluble ligands

Aromatic amino acids are believed to play a pivotal role in carbohydrate-bi nding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM10s ), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan re sidues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens sub sp, cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the muta nt proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with th e glucose polymer. When the W7A mutation was introduced into CBM10 the prot ein domain did not accumulate in Escherichia coli. In contrast, the W7A mut ant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W 24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of th e protein, while Trp7 was buried. Collectively, these data indicate that Tr p22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 t o cellulose. The results are discussed in the light of the three-dimensiona l structure of CBM10 [Raghothama, S., Simpson, P. J., Szabo, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984].