4KZF

The mechanism of the amidases: The effect of the mutation E142L in the amidase from Geobacillus pallidus


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.203 
  • R-Value Work: 0.185 
  • R-Value Observed: 0.186 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning.

Weber, B.W.Kimani, S.W.Varsani, A.Cowan, D.A.Hunter, R.Venter, G.A.Gumbart, J.C.Sewell, B.T.

(2013) J Biol Chem 288: 28514-28523

  • DOI: https://doi.org/10.1074/jbc.M113.503284
  • Primary Citation of Related Structures:  
    4GYL, 4GYN, 4KZF, 4LF0

  • PubMed Abstract: 

    All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys "catalytic triad." In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained despite the mutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu-59) in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O(ε1) in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O(δ1.) In neither case is an atom located at the position of Glu-142 O(ε2) in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O(ε2) and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.


  • Organizational Affiliation

    From the Structural Biology Research Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences and.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Aliphatic amidase348Bacillus sp. (in: firmicutes)Mutation(s): 1 
Gene Names: amiEami
EC: 3.5.1.4
UniProt
Find proteins for Q9L543 (Bacillus sp)
Explore Q9L543 
Go to UniProtKB:  Q9L543
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9L543
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CL
Query on CL

Download Ideal Coordinates CCD File 
B [auth A]CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.203 
  • R-Value Work: 0.185 
  • R-Value Observed: 0.186 
  • Space Group: P 42 3 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 130.979α = 90
b = 130.979β = 90
c = 130.979γ = 90
Software Package:
Software NamePurpose
XSCALEdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
MxCuBEdata collection
d*TREKdata reduction
d*TREKdata scaling
REFMACphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-08-21
    Type: Initial release
  • Version 1.1: 2014-02-12
    Changes: Database references
  • Version 1.2: 2023-11-08
    Changes: Data collection, Database references, Derived calculations, Refinement description