RCSB PDB - 8F5W: Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine and NADPH Quasi-Anaerobically

 8F5W

Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine and NADPH Quasi-Anaerobically


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.97 Å
  • R-Value Free: 0.217 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 

Starting Model: experimental
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wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted FADClick on this verticalbar to view detailsBest fitted NAPClick on this verticalbar to view detailsBest fitted FNRClick on this verticalbar to view detailsBest fitted TDRClick on this verticalbar to view details

This is version 1.2 of the entry. See complete history


Literature

Mammalian dihydropyrimidine dehydrogenase: Added mechanistic details from transient-state analysis of charge transfer complexes.

Smith, M.M.Forouzesh, D.C.Kaley, N.E.Liu, D.Moran, G.R.

(2023) Arch Biochem Biophys 736: 109517-109517

  • DOI: https://doi.org/10.1016/j.abb.2023.109517
  • Primary Citation of Related Structures:  
    8F5W, 8F61, 8F6N

  • PubMed Abstract: 

    Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by ∼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe 4 S 4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe 4 S 4 centers to yield the active state with a cofactor set of FAD•4(Fe 4 S 4 )•FMNH 2 . The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP + indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzyme•pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP + DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.


  • Organizational Affiliation

    Department of Chemistry and Biochemistry, 1068 W Sheridan Rd, Loyola University Chicago, Chicago, IL, 60660, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Dihydropyrimidine dehydrogenase [NADP(+)]
A, B, C, D
1,025Sus scrofaMutation(s): 1 
Gene Names: DPYD
EC: 1.3.1.2
UniProt
Find proteins for Q28943 (Sus scrofa)
Explore Q28943 
Go to UniProtKB:  Q28943
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ28943
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 5 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
FAD (Subject of Investigation/LOI)
Query on FAD

Download Ideal Coordinates CCD File 
GA [auth D],
I [auth A],
Q [auth B],
Y [auth C]
FLAVIN-ADENINE DINUCLEOTIDE
C27 H33 N9 O15 P2
VWWQXMAJTJZDQX-UYBVJOGSSA-N
NAP (Subject of Investigation/LOI)
Query on NAP

Download Ideal Coordinates CCD File 
HA [auth D],
J [auth A],
R [auth B],
Z [auth C]
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
C21 H28 N7 O17 P3
XJLXINKUBYWONI-NNYOXOHSSA-N
FNR (Subject of Investigation/LOI)
Query on FNR

Download Ideal Coordinates CCD File 
AA [auth C],
IA [auth D],
K [auth A],
T [auth B]
1-DEOXY-1-(7,8-DIMETHYL-2,4-DIOXO-3,4-DIHYDRO-2H-BENZO[G]PTERIDIN-1-ID-10(5H)-YL)-5-O-PHOSPHONATO-D-RIBITOL
C17 H23 N4 O9 P
YTNIXZGTHTVJBW-SCRDCRAPSA-N
SF4 (Subject of Investigation/LOI)
Query on SF4

Download Ideal Coordinates CCD File 
CA [auth D]
DA [auth D]
E [auth A]
EA [auth D]
F [auth A]
IRON/SULFUR CLUSTER
Fe4 S4
LJBDFODJNLIPKO-UHFFFAOYSA-N
TDR (Subject of Investigation/LOI)
Query on TDR

Download Ideal Coordinates CCD File 
BA [auth C],
JA [auth D],
L [auth A],
S [auth B]
THYMINE
C5 H6 N2 O2
RWQNBRDOKXIBIV-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.97 Å
  • R-Value Free: 0.217 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 81.735α = 90
b = 158.003β = 96.06
c = 162.404γ = 90
Software Package:
Software NamePurpose
xia2data reduction
xia2data scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACTdata extraction

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted FADClick on this verticalbar to view detailsBest fitted NAPClick on this verticalbar to view detailsBest fitted FNRClick on this verticalbar to view detailsBest fitted TDRClick on this verticalbar to view details

Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Science Foundation (NSF, United States)United States1904480
National Science Foundation (NSF, United States)United States2203593

Revision History  (Full details and data files)

  • Version 1.0: 2023-02-01
    Type: Initial release
  • Version 1.1: 2023-02-08
    Changes: Database references
  • Version 1.2: 2023-10-25
    Changes: Data collection, Refinement description