1Y7A

Structure of D153H/K328W E. coli alkaline phosphatase in presence of cobalt at 1.77 A resolution


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.77 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Metal Specificity Is Correlated with Two Crucial Active Site Residues in Escherichia coli Alkaline Phosphatase(,).

Wang, J.Stieglitz, K.A.Kantrowitz, E.R.

(2005) Biochemistry 44: 8378-8386

  • DOI: https://doi.org/10.1021/bi050155p
  • Primary Citation of Related Structures:  
    1Y6V, 1Y7A

  • PubMed Abstract: 

    Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.


  • Organizational Affiliation

    Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alkaline phosphatase
A, B
449Escherichia coliMutation(s): 2 
Gene Names: phoA
EC: 3.1.3.1
UniProt
Find proteins for P00634 (Escherichia coli (strain K12))
Explore P00634 
Go to UniProtKB:  P00634
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00634
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
G [auth A],
L [auth B]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
PO4
Query on PO4

Download Ideal Coordinates CCD File 
F [auth A],
K [auth B]
PHOSPHATE ION
O4 P
NBIIXXVUZAFLBC-UHFFFAOYSA-K
CO
Query on CO

Download Ideal Coordinates CCD File 
C [auth A]
D [auth A]
E [auth A]
H [auth B]
I [auth B]
C [auth A],
D [auth A],
E [auth A],
H [auth B],
I [auth B],
J [auth B]
COBALT (II) ION
Co
XLJKHNWPARRRJB-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.77 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 
  • Space Group: I 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 76.208α = 90
b = 164.564β = 90
c = 192.636γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
SCALEPACKdata scaling
AMoREphasing
CNSrefinement
HKL-2000data reduction

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-06-21
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-23
    Changes: Data collection, Refinement description