3FYO

Crystal structure of the triple mutant (N23C/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.216 
  • R-Value Observed: 0.218 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Reversing evolution: re-establishing obligate metal ion dependence in a metal-independent KDO8P synthase

Cochrane, F.C.Cookson, T.V.Jameson, G.B.Parker, E.J.

(2009) J Mol Biol 390: 646-661

  • DOI: https://doi.org/10.1016/j.jmb.2009.05.014
  • Primary Citation of Related Structures:  
    3FYO, 3FYP

  • PubMed Abstract: 

    Two distinct groups of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved (246)CysAspGlyPro(249) motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd(2+), the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 A. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu(2+). Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.


  • Organizational Affiliation

    Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
A, B, C, D
280Neisseria meningitidis serogroup BMutation(s): 3 
Gene Names: kdsANMB1283
EC: 2.5.1.55
UniProt
Find proteins for Q9JZ55 (Neisseria meningitidis serogroup B (strain MC58))
Explore Q9JZ55 
Go to UniProtKB:  Q9JZ55
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9JZ55
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.216 
  • R-Value Observed: 0.218 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 82.74α = 90
b = 86.197β = 90
c = 163.891γ = 90
Software Package:
Software NamePurpose
d*TREKdata scaling
AMoREphasing
REFMACrefinement
PDB_EXTRACTdata extraction
CrystalCleardata collection
d*TREKdata reduction

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-03-24
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Advisory, Refinement description, Version format compliance
  • Version 1.2: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.3: 2023-09-06
    Changes: Data collection, Refinement description