A single residue mutation abolishes attachment of the CBM26 starch-binding domain from Lactobacillus amylovorus alpha-amylase.
Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus alpha-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus alpha-amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each alpha-amylase SBD.
Rodríguez-Sanoja
Romina
R
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, UNAM, A. P. 70228, Ciudad Universitaria, 04510, México D.F., Mexico romina@correo.biomedicas.unam.mx
Oviedo
N
N
Escalante
L
L
Ruiz
B
B
Sánchez
S
S
D000595:Amino Acid Sequence; D007779:Lactobacillus acidophilus / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D013213:Starch; D000516:alpha-Amylases