3B8T

Crystal structure of Escherichia coli alaine racemase mutant P219A


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.00 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.219 

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Ligand Structure Quality Assessment 


This is version 1.4 of the entry. See complete history


Literature

Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

Wu, D.Hu, T.Zhang, L.Chen, J.Du, J.Ding, J.Jiang, H.Shen, X.

(2008) Protein Sci 17: 1066-1076

  • DOI: https://doi.org/10.1110/ps.083495908
  • Primary Citation of Related Structures:  
    2RJG, 2RJH, 3B8T, 3B8U, 3B8V, 3B8W

  • PubMed Abstract: 

    Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.


  • Organizational Affiliation

    Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alanine racemase
A, B, C, D
379Escherichia coli K-12Mutation(s): 1 
Gene Names: alr
EC: 5.1.1.1
UniProt
Find proteins for P0A6B4 (Escherichia coli (strain K12))
Explore P0A6B4 
Go to UniProtKB:  P0A6B4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A6B4
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
PLP
Query on PLP

Download Ideal Coordinates CCD File 
H [auth A],
K [auth B],
N [auth C],
O [auth D]
PYRIDOXAL-5'-PHOSPHATE
C8 H10 N O6 P
NGVDGCNFYWLIFO-UHFFFAOYSA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
E [auth A]
F [auth A]
G [auth A]
I [auth B]
J [auth B]
E [auth A],
F [auth A],
G [auth A],
I [auth B],
J [auth B],
L [auth C],
M [auth C]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
KCX
Query on KCX
A, B, C, D
L-PEPTIDE LINKINGC7 H14 N2 O4LYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.00 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.219 
  • Space Group: P 6
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 147.921α = 90
b = 147.921β = 90
c = 163.797γ = 120
Software Package:
Software NamePurpose
MOSFLMdata reduction
SCALAdata scaling
MOLREPphasing
CNSrefinement
PDB_EXTRACTdata extraction
CrystalCleardata collection
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: 2008-07-08
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Non-polymer description, Version format compliance
  • Version 1.2: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.3: 2023-11-01
    Changes: Data collection, Refinement description
  • Version 1.4: 2023-11-15
    Changes: Data collection