3LIH

Crystal structure of fructosyltransferase (D191A) from A. japonicus in complex with raffinose


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.222 
  • R-Value Observed: 0.227 

wwPDB Validation   3D Report Full Report


This is version 2.1 of the entry. See complete history


Literature

Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis

Chuankhayan, P.Hsieh, C.Y.Huang, Y.C.Hsieh, Y.Y.Guan, H.H.Hsieh, Y.C.Tien, Y.C.Chen, C.D.Chiang, C.M.Chen, C.J.

(2010) J Biol Chem 285: 23251-23264

  • DOI: https://doi.org/10.1074/jbc.M110.113027
  • Primary Citation of Related Structures:  
    3LIH

  • PubMed Abstract: 

    Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.


  • Organizational Affiliation

    Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Fructosyltransferase634Aspergillus japonicusMutation(s): 0 
UniProt
Find proteins for Q1W3Z7 (Aspergillus japonicus)
Explore Q1W3Z7 
Go to UniProtKB:  Q1W3Z7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ1W3Z7
Sequence Annotations
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  • Reference Sequence
Oligosaccharides

Help

Entity ID: 2
MoleculeChains Length2D Diagram Glycosylation3D Interactions
alpha-D-galactopyranose-(1-6)-alpha-D-glucopyranose-(1-2)-beta-D-fructofuranose
B
3N/A
Glycosylation Resources
GlyTouCan:  G40178VU
GlyCosmos:  G40178VU
Biologically Interesting Molecules (External Reference) 1 Unique
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.222 
  • R-Value Observed: 0.227 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 99.268α = 90
b = 109.938β = 90
c = 65.785γ = 90
Software Package:
Software NamePurpose
ADSCdata collection
CNSrefinement
HKL-2000data reduction
HKL-2000data scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-05-12
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2012-05-30
    Changes: Database references, Non-polymer description
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Atomic model, Data collection, Derived calculations, Non-polymer description, Structure summary
  • Version 2.1: 2024-03-20
    Changes: Data collection, Database references, Structure summary