1JD9

CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.180 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Structural basis of alpha-amylase activation by chloride

Aghajari, N.Feller, G.Gerday, C.Haser, R.

(2002) Protein Sci 11: 1435-1441

  • DOI: https://doi.org/10.1110/ps.0202602
  • Primary Citation of Related Structures:  
    1JD7, 1JD9, 1L0P

  • PubMed Abstract: 

    To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.


  • Organizational Affiliation

    Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS-UCBL1, Laboratoire de Bio-Cristallographie, 69367 Lyon, France.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
ALPHA-AMYLASE453Pseudoalteromonas haloplanktisMutation(s): 1 
Gene Names: AMY
EC: 3.2.1.1
UniProt
Find proteins for P29957 (Pseudoalteromonas haloplanktis)
Explore P29957 
Go to UniProtKB:  P29957
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP29957
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CA
Query on CA

Download Ideal Coordinates CCD File 
B [auth A]CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.180 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 71.5α = 90
b = 138.4β = 90
c = 115.4γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALAdata scaling
X-PLORmodel building
X-PLORrefinement
CCP4data scaling
X-PLORphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-09-18
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-10-25
    Changes: Data collection, Refinement description