FIG. 3.

Site-directed mutational analysis of conserved large aromatic residues in domain XX, in order to determine residues important for carbohydrate binding. (A) Amino acid sequence alignments of the FSUAXH1 XX domain and its homologous peptides. The amino acid sequences were obtained through an NCBI BLAST search and aligned with that of domain XX of FSUAXH1 by utilizing ClustalW. The conserved tyrosines (Y) and tryptophans (W) are indicated by arrows. The amino acid numbers are based on the numbering of the FSUAXH1 polypeptide. The NCBI accession numbers (source of organism) were as follows: YP_001181179 (Caldicellulosiruptor saccharolyticus DSM 8903), YP_002572107 (Anaerocellum thermophilum DSM 6725), ZP_02421660 (Eubacterium siraeum DSM 15702), ZP_03678241 (Bacteroides cellulosilyticus DSM 14838), ZP_03013018 (Bacteroides intestinalis DSM 17393), ZP_03458529 (Bacteroides eggerthii DSM 20697), YP_001196205 (Flavobacterium johnsoniae UW101), YP_678646 (Cytophaga hutchinsonii ATCC 33406), and YP_003012971 (Paenibacillus sp. JDR-2). The output files were entered into the BoxShade version 3.21 program (http://www.ch.embnet.org/software/BOX_form.html), with the fraction of sequences that must agree for shading set at 1.0. The conserved amino acids are shaded black, and similar amino acids are shaded gray. (B) Binding assay for FSUAXH1 TM2 and its site-directed mutant proteins. The proteins (1 μg/lane) were resolved on a nondenaturing 5% polyacrylamide gel containing 0% (control) or 0.1% (wt/vol) arabinoxylan and stained with Coomassie brilliant blue G-250.