1H7K

Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.240 
  • R-Value Work: 0.237 
  • R-Value Observed: 0.237 

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This is version 1.4 of the entry. See complete history


Literature

Formation of a Tyrosyl Radical Intermediate in Proteus Mirabilis Catalase by Directed Mutagenesis and Consequences for Nucleotide Reactivity.

Andreoletti, P.Gambarelli, S.Sainz, G.Stojanoff, V.White, C.Desfonds, G.Gagnon, J.Gaillard, J.Jouve, H.M.

(2001) Biochemistry 40: 13734

  • DOI: https://doi.org/10.1021/bi010687f
  • Primary Citation of Related Structures:  
    1H7K

  • PubMed Abstract: 

    Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding catalases. The function of NADPH in these enzymes is still a matter of debate. This study presents the effects of two independent phenylalanine mutations (F194 and F215), located between NADPH and heme in the PMC structure. The phenylalanines were replaced with tyrosines which we predicted could carry radicals in a NADPH-heme electron transfer. The X-ray crystal structures of the two mutants indicated that neither the binding site of NADPH nor the immediate environment of the residues was affected by the mutations. Measurements using H2O2 as a substrate confirmed that the variants were as active as the native enzyme. With equivalent amounts of peroxoacetic acid, wild-type PMC, F215Y PMC, and beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC variant produced a compound I which was rapidly transformed into compound II and a tyrosyl radical. EPR studies showed that this radical, generated by the oxidation of Y194, was not related to the previously observed radical in BLC, located on Y369. In the presence of excess NADPH, compound I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron process. This was independent of the enzyme's origin and did not require any thus far identified tyrosyl radicals. Conversely, the presence of a tyrosyl radical in F194Y PMC greatly enhanced the oxidation of reduced beta-nicotinamide mononucleotide under a steady-state H2O2 flow with observable compound II. This process could involve a one-electron reduction of compound I via Y194.


  • Organizational Affiliation

    Institut de Biologie Structurale Jean-Pierre Ebel, CEA/CNRS/UJF, UMR 5075, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CATALASE483Proteus mirabilisMutation(s): 1 
EC: 1.11.1.6
UniProt
Find proteins for P42321 (Proteus mirabilis)
Explore P42321 
Go to UniProtKB:  P42321
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP42321
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
OMT
Query on OMT
A
L-PEPTIDE LINKINGC5 H11 N O4 SMET
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.240 
  • R-Value Work: 0.237 
  • R-Value Observed: 0.237 
  • Space Group: P 62 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 110α = 90
b = 110β = 90
c = 251.2γ = 120
Software Package:
Software NamePurpose
CNSrefinement
MOSFLMdata reduction
SCALAdata scaling
CNSphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-02-25
    Type: Initial release
  • Version 1.1: 2015-09-16
    Changes: Database references, Derived calculations, Non-polymer description, Other, Refinement description, Source and taxonomy, Structure summary, Version format compliance
  • Version 1.2: 2019-04-03
    Changes: Data collection, Derived calculations, Experimental preparation, Other
  • Version 1.3: 2019-05-08
    Changes: Data collection, Experimental preparation
  • Version 1.4: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description