The α-galactosidase AgaA from the thermophilic microorganism Geobacillus stearothermophilus has great industrial potential because it is fully active at 338 K against raffinose and can increase the yield of manufactured sucrose. AgaB has lower affinity for its natural substrates but is a powerful tool for the enzymatic synthesis of disaccharides by transglycosylation. These two enzymes have 97% identity and belong to the glycoside hydrolase (GH) family GH36, for which few structures are available. To understand the structural basis underlying the differences between these two enzymes, we determined the crystal structures of AgaA and AgaB by molecular replacement at 3.2 Å and 1.8 Å resolution, respectively. We also solved a 2.8 Å structure of the AgaAA355E mutant, which has enzymatic properties similar to those of AgaB. We observe that residue 355 is located 20 Å away from the active site and that the A355E substitution causes structural rearrangements resulting in a significant displacement of the invariant Trp-336 at catalytic subsite -1. Hence, the active cleft of AgaA is narrowed in comparison to AgaB, and AgaA is more efficient than AgaB against its natural substrates. The structure of AgaAA355E, complexed with 1-deoxygalactonojirimycin, reveals an induced fit movement; there is a rupture of the electrostatic interaction between Glu-355 and Asn-335 and a return of Trp-336 to an optimal position for ligand stacking. The structures of two catalytic mutants of AgaAA355E complexed with raffinose and stachyose show that the binding interactions are stronger at subsite -1 to enable the binding of various α-galactosides.
The article is published online in the journal of Biological Chemistry and is free to access.