4FNU

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


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.60 Å
  • R-Value Free: 0.282 
  • R-Value Work: 0.238 
  • R-Value Observed: 0.240 

wwPDB Validation   3D Report Full Report


This is version 2.1 of the entry. See complete history


Literature

The molecular mechanism of the thermostable alpha-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies

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

(2012) J Biol Chem 287: 39642-39652

  • DOI: https://doi.org/10.1074/jbc.M112.394114
  • Primary Citation of Related Structures:  
    4FNP, 4FNQ, 4FNR, 4FNS, 4FNT, 4FNU

  • 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.


  • Organizational Affiliation

    Biocrystallography and Structural Biology of Therapeutic Targets, Bases Moléculaires et Structurales des Systèmes Infectieux-Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS, Université de Lyon 1, 7 passage du Vercors, 69367 Lyon Cedex 07, France.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alpha-galactosidase AgaA
A, B, C, D
729Geobacillus stearothermophilusMutation(s): 2 
Gene Names: agaA
EC: 3.2.1.22
UniProt
Find proteins for Q9ALJ4 (Geobacillus stearothermophilus)
Explore Q9ALJ4 
Go to UniProtKB:  Q9ALJ4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9ALJ4
Sequence Annotations
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  • Reference Sequence
Oligosaccharides

Help

Entity ID: 2
MoleculeChains Length2D Diagram Glycosylation3D Interactions
beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose-(1-6)-alpha-D-galactopyranose-(1-6)-alpha-D-galactopyranose
E, F, G, H
4N/A
Glycosylation Resources
GlyTouCan:  G21073BE
GlyCosmos:  G21073BE
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.60 Å
  • R-Value Free: 0.282 
  • R-Value Work: 0.238 
  • R-Value Observed: 0.240 
  • Space Group: P 32 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 154.07α = 90
b = 154.07β = 90
c = 238.02γ = 120
Software Package:
Software NamePurpose
ADSCdata collection
MOLREPphasing
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-10-03
    Type: Initial release
  • Version 1.1: 2013-02-20
    Changes: Database references
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Atomic model, Data collection, Database references, Derived calculations, Structure summary
  • Version 2.1: 2024-02-28
    Changes: Data collection, Database references, Structure summary