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Structure of IDP01828

2.0 Angstrom Crystal structure of Glutamate-Cysteine Ligase (gshA) ftom Francisella tularensis in Comlex with AMP.

Edit deposit information
CSGID target
IDP01828 
PDB Id
3NZT (NCBI MMDB
Authors
G.Minasov,A.Halavaty,L.Shuvalova,I.Dubrovska,J.Winsor,L.Papazisi,W.F.Anderson,Center For Structural Genomics Of Infectious Diseases (Csgid) 
Responsible person
George Minasov 
Responsible lab
Northwestern University 
Deposition Date
Jul 16, 2010 
Release Date
Jul 28, 2010 

Annotation

Description
The IDP01828 glutamate-cysteine ligase from Francisella tularensis subsp. tularensis SCHU S4 is a rate-limiting enzyme that participates in the glutamate and glutathione metabolism and is a target for development of potential therapeutic agents against parasites and cancer. The catalytic mechanism of the enzyme has been proposed to involve the initial activation of the γ-carboxyl group of L-Glu by ATP-phosphorylation to form a γ-glutamylphosphate intermediate, followed by the nucleophilic attack of the amino group of L-Cys on the carbonyl to generate a tetrahedral transition state. Thus, the enzyme utilizes three substrates, ATP, L-Glu and L-Cys. The IDP01828 protein was initially co-crystallized with the ATP analog, 1mM AMPPNP, in the presence of 1mM MgCl2, however, the final crystal structure (PDB ID code 3NZT) contains only AMP part of the ligand in the active site. The nucleoside part of AMP sits in a deep groove at a wider opening of a 29 A long negatively charged tunnel, which goes through the entire protein. Two longest beta strands (residues ranges 10-21 and 136-141) of the 47 % helical tangled ligase form a base of this depression. The phosphate group of the nucleotide points toward the positively charged pocket at the beginning of the wider opening of the tunnel. Four sulfate ions have been modeled inside the tunnel in the vicinity of where two other substrates, L-Glu and L-Cys, shall bind. The opposite end of the tunnel is half way closed with a Asp218-Lys227 salt bridge, which might also locally stabilize the tertiary structure of the protein.  
Functional assignment
Ligase 

Ligands

Ligand code Name Ligand type
SO4
AMP

Structure information

Unit cell parameters

Space Group
P 65  
Unit Cell

a=89.85Å, b=89.85Å, c=151.47Å
α=90.00, β=90.00, γ=120.00 
Solvent content
58.38  
Matthews coefficient
2.96  

Refinement

Data for the highest resolution shell is in parentheses.
Resolution range
29.41-2.00Å (2.05-2.00Å)  
Rall(%)
16.5 
Rwork(%)
16.4 (20.5) 
Rfree(%)
19.6 (23.0) 
Num. observed reflections
46557 (3404) 
Num. Rfree reflections
2327 (182) 
Completeness(%)
99.9 (99.4) 

Model parameters

Num Atoms
4530  
Num Waters
328  
Num Hetatoms
0  
Model mean isotropic B factor
29.230Å2  
RMSD bond length
0.012Å  
RMSD bond angle
1.372°  
Filename uploaded
idp01828f-deposit.pdb (uploaded on Jul 16, 2010 9:15 PM)  
Inserted
Jul 16, 2010