Abstract
Alpha-galactosidases catalyze the hydrolysis of terminal alpha-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae alpha-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 A resolution. The monomer folds into a catalytic (alpha/beta)(8) barrel and a C-terminal beta-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the beta-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.
Highlights
Glycosyl hydrolases are classified into 115 different families in the CAZy data base according to their amino acid similarities [8]
We report here the structure of the Saccharomyces cerevisiae ␣-galactosidase (ScAGal) at 1.95 Å and the structure of the complexes with melibiose and raffinose at 2.4 and 2.7 Å resolution, respectively
We address here some structural features of ScAGal and other GH27 enzymes regarding substrate recognition and specificity, oligomeric state, and protein stability that remained unexplained
Summary
Cloning and Mutagenesis—The MEL1 gene (GB X03102) encoding an ␣-galactosidase from S. cerevisiae (ScAGal; Uniprot P04824) was amplified and cloned into YEpFLAG-1 vector (Eastman Kodak Co.) as described previously [10]. Crystallization and Data Collection—Crystallization of ScAGal (2.5 mg mlϪ1 in 0.05 M Tris-HCl, pH 7.4, 0.150 M NaCl, and 0.002 M DTT) and mutant D149A-ScAGal (1.8 mg mlϪ1 in 0.05 M Tris-HCl, pH 7.4, 0.150 M NaCl, and 0.002 M DTT) was performed on Cryschem (Hampton Research) sitting drop plates at 291 K as described previously [10]. The structure of Oryza sativa (rice) ␣-galactosidase (Protein Data Bank code 1UAS) [15] was used to prepare the search model using the program CHAINSAW [16], and the ScAGal sequence was aligned to that from rice ␣-galactosidase. The structures of the complexes were solved by molecular replacement with the native model, and refinement was performed as described above. The protein model contained the coordinates of the tetrameric ScAGal structure presented here, after removing all nonpolypeptide atoms. Kinetic characterization of ScAGal and mutants was performed assaying ␣-galactosidase activity of purified protein samples (described above) toward different substrate concentrations. The weight average molecular weight was determined by using the program EQASSOC with the partial specific volume of ScAGal set to 0.72 at 293 K as calculated from its amino acid composition
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