4DQK

Crystal structure of the FAD binding domain of cytochrome P450 BM3


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.256 
  • R-Value Work: 0.213 
  • R-Value Observed: 0.215 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

The crystal structure of the FAD/NADPH-binding domain of flavocytochrome P450 BM3.

Joyce, M.G.Ekanem, I.S.Roitel, O.Dunford, A.J.Neeli, R.Girvan, H.M.Baker, G.J.Curtis, R.A.Munro, A.W.Leys, D.

(2012) FEBS J 279: 1694-1706

  • DOI: https://doi.org/10.1111/j.1742-4658.2012.08544.x
  • Primary Citation of Related Structures:  
    4DQK, 4DQL

  • PubMed Abstract: 

    We report the crystal structure of the FAD/NADPH-binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP(+), identifying important protein interactions with the NADPH 2'-phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH-dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild-type FAD domain to be near-exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme's connecting domain (that links the FAD domain to the FMN-binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module.


  • Organizational Affiliation

    Department of Biochemistry, University of Leicester, Leicester, UK.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Bifunctional P-450/NADPH-P450 reductase
A, B
391Priestia megateriumMutation(s): 1 
Gene Names: CYP102A1cyp102
EC: 1.14.14.1 (PDB Primary Data), 1.6.2.4 (PDB Primary Data)
UniProt
Find proteins for P14779 (Priestia megaterium (strain ATCC 14581 / DSM 32 / CCUG 1817 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / NRRL B-14308 / VKM B-512 / Ford 19))
Explore P14779 
Go to UniProtKB:  P14779
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP14779
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
FAD
Query on FAD

Download Ideal Coordinates CCD File 
C [auth A],
I [auth B]
FLAVIN-ADENINE DINUCLEOTIDE
C27 H33 N9 O15 P2
VWWQXMAJTJZDQX-UYBVJOGSSA-N
PG4
Query on PG4

Download Ideal Coordinates CCD File 
G [auth A],
H [auth A],
M [auth B],
N [auth B]
TETRAETHYLENE GLYCOL
C8 H18 O5
UWHCKJMYHZGTIT-UHFFFAOYSA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
D [auth A]
E [auth A]
F [auth A]
J [auth B]
K [auth B]
D [auth A],
E [auth A],
F [auth A],
J [auth B],
K [auth B],
L [auth B]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.256 
  • R-Value Work: 0.213 
  • R-Value Observed: 0.215 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 191.387α = 90
b = 191.387β = 90
c = 74.211γ = 120
Software Package:
Software NamePurpose
HKL-2000data collection
PHASERphasing
REFMACrefinement
HKL-3000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-03-07
    Type: Initial release
  • Version 1.1: 2012-06-06
    Changes: Database references
  • Version 1.2: 2014-11-12
    Changes: Structure summary
  • Version 1.3: 2024-02-28
    Changes: Data collection, Database references, Derived calculations