2GLX

Crystal Structure Analysis of bacterial 1,5-AF Reductase


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.193 

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Ligand Structure Quality Assessment 


This is version 1.4 of the entry. See complete history


Literature

Crystal Structure of NADP(H)-Dependent 1,5-Anhydro-d-fructose Reductase from Sinorhizobium morelense at 2.2 A Resolution: Construction of a NADH-Accepting Mutant and Its Application in Rare Sugar Synthesis

Dambe, T.R.Kuehn, A.M.Brossette, T.Giffhorn, F.Scheidig, A.J.

(2006) Biochemistry 45: 10030-10042

  • DOI: https://doi.org/10.1021/bi052589q
  • Primary Citation of Related Structures:  
    2GLX

  • PubMed Abstract: 

    Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.


  • Organizational Affiliation

    Abteilung Strukturbiologie, Fachbereich Biophysik, Universitätsklinikum des Saarlandes, D-66424 Homburg/Saar, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
1,5-anhydro-D-fructose reductase
A, B, C, D, E
A, B, C, D, E, F
332Ensifer adhaerensMutation(s): 0 
Gene Names: afr
EC: 1.1.1.263
UniProt
Find proteins for Q2I8V6 (Ensifer adhaerens)
Explore Q2I8V6 
Go to UniProtKB:  Q2I8V6
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ2I8V6
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NDP
Query on NDP

Download Ideal Coordinates CCD File 
H [auth A]
J [auth B]
L [auth C]
N [auth D]
P [auth E]
H [auth A],
J [auth B],
L [auth C],
N [auth D],
P [auth E],
R [auth F]
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
C21 H30 N7 O17 P3
ACFIXJIJDZMPPO-NNYOXOHSSA-N
ACT
Query on ACT

Download Ideal Coordinates CCD File 
G [auth A]
I [auth B]
K [auth C]
M [auth D]
O [auth E]
G [auth A],
I [auth B],
K [auth C],
M [auth D],
O [auth E],
Q [auth F]
ACETATE ION
C2 H3 O2
QTBSBXVTEAMEQO-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.193 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 97.19α = 90
b = 84.9β = 96.3
c = 150.94γ = 90
Software Package:
Software NamePurpose
XSCALEdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
ProDCdata collection
SOLVEphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2006-08-29
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-10-18
    Changes: Refinement description
  • Version 1.4: 2024-03-13
    Changes: Data collection, Database references, Derived calculations