1W5O

Stepwise introduction of zinc binding site into porphobilinogen synthase of Pseudomonas aeruginosa (mutations A129C, D131C and D139C)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.176 
  • R-Value Work: 0.131 
  • R-Value Observed: 0.134 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro

Frere, F.Reents, H.Schubert, W.-D.Heinz, D.W.Jahn, D.

(2005) J Mol Biol 345: 1059

  • DOI: https://doi.org/10.1016/j.jmb.2004.10.053
  • Primary Citation of Related Structures:  
    1W54, 1W56, 1W5M, 1W5N, 1W5O, 1W5P, 1W5Q

  • PubMed Abstract: 

    Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.


  • Organizational Affiliation

    Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DELTA-AMINOLEVULINIC ACID DEHYDRATASE
A, B
337Pseudomonas aeruginosa PAO1Mutation(s): 3 
EC: 4.2.1.24
UniProt
Find proteins for Q59643 (Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1))
Explore Q59643 
Go to UniProtKB:  Q59643
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ59643
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
GOL
Query on GOL

Download Ideal Coordinates CCD File 
C [auth A],
G [auth B]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
ZN
Query on ZN

Download Ideal Coordinates CCD File 
F [auth A],
J [auth B]
ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
K
Query on K

Download Ideal Coordinates CCD File 
D [auth A],
H [auth B]
POTASSIUM ION
K
NPYPAHLBTDXSSS-UHFFFAOYSA-N
MG
Query on MG

Download Ideal Coordinates CCD File 
E [auth A],
I [auth B]
MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.176 
  • R-Value Work: 0.131 
  • R-Value Observed: 0.134 
  • Space Group: P 4 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 126.991α = 90
b = 126.991β = 90
c = 85.369γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-01-19
    Type: Initial release
  • Version 1.1: 2011-05-08
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2019-05-08
    Changes: Data collection, Experimental preparation, Other
  • Version 1.4: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description