4FNU

Crystal structure of GH36 alpha-galactosidase AgaA A355E D478A from Geobacillus stearothermophilus in complex with stachyose

Summary for 4FNU

• Entry DOI: 10.2210/pdb4fnu/pdb
• Descriptor: Alpha-galactosidase AgaA, beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose-(1-6)-alpha-D-galactopyranose-(1-6)-alpha-D-galactopyranose (2 entities in total)
• Functional Keywords: glycoside hydrolase, hydrolase, carbohydrate
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 4
• Total formula weight: 335954.03

Authors

Merceron, R.,Foucault, M.,Haser, R.,Mattes, R.,Watzlawick, H.,Gouet, P. (deposition date: 2012-06-20, release date: 2012-10-03, Last modification date: 2024-02-28)

Primary citation

Merceron, R.,Foucault, M.,Haser, R.,Mattes, R.,Watzlawick, H.,Gouet, P.
The molecular mechanism of the thermostable alpha-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies
J.Biol.Chem., 287:39642-39652, 2012

PubMed Abstract: 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- and 1.8 Å-resolution, respectively. We also solved a 2.8-Å structure of the AgaA(A355E) 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 with AgaB, and AgaA is more efficient than AgaB against its natural substrates. The structure of AgaA(A355E) 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 AgaA(A355E) complexed with raffinose and stachyose show that the binding interactions are stronger at subsite -1 to enable the binding of various α-galactosides.

PubMed: 23012371
DOI: 10.1074/jbc.M112.394114

Experimental method

X-RAY DIFFRACTION (3.6 Å)