PDBsum entry 4fns

Hydrolase

PDB id: 4fns

Contents

Protein chains

718 a.a.

Ligands

DGJ ×4

SO4 ×8

EDO

Waters ×980

PDB id:

4fns

Name:

Hydrolase

Title:

Crystal structure of gh36 alpha-galactosidase agaa a355e from geobacillus stearothermophilus in complex with 1- deoxygalactonojirimycin

Structure:

Alpha-galactosidase agaa. Chain: a, b, c, d. Engineered: yes. Mutation: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Gene: agaa. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.60Å R-factor: 0.177 R-free: 0.231

Authors:

R.Merceron,M.Foucault,R.Haser,R.Mattes,H.Watzlawick,P.Gouet

Key ref:

R.Merceron et al. (2012). The molecular mechanism of thermostable α-galactosidases AgaA and AgaB explained by x-ray crystallography and mutational studies. J Biol Chem, 287, 39642-39652. PubMed id: 23012371

Date:

20-Jun-12 Release date: 03-Oct-12

Protein chains

Q9ALJ4  (AGAA_GEOSE) -  Alpha-galactosidase AgaA from Geobacillus stearothermophilus

Seq: 729 a.a.

Struc: 718 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.22 - alpha-galactosidase.
• Reaction: Melibiose + H₂O = galactose + glucose
• Cofactor: Mg(2+); NAD(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

J Biol Chem 287:39642-39652 (2012)

PubMed id: 23012371

The molecular mechanism of thermostable α-galactosidases AgaA and AgaB explained by x-ray crystallography and mutational studies.

R.Merceron, M.Foucault, R.Haser, R.Mattes, H.Watzlawick, P.Gouet.

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.