RCSB PDB - 3KSD: Crystal Structure of C151S+H178N mutant of Glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1) from Staphylococcus aureus MRSA252 complexed with NAD at 2.2 angstrom resolution

 3KSD

Crystal Structure of C151S+H178N mutant of Glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1) from Staphylococcus aureus MRSA252 complexed with NAD at 2.2 angstrom resolution


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.208 

Starting Model: experimental
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Literature

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase 1 from methicillin-resistant Staphylococcus aureus MRSA252 provides novel insights into substrate binding and catalytic mechanism.

Mukherjee, S.Dutta, D.Saha, B.Das, A.K.

(2010) J Mol Biol 401: 949-968

  • DOI: https://doi.org/10.1016/j.jmb.2010.07.002
  • Primary Citation of Related Structures:  
    3HQ4, 3K73, 3K9Q, 3KSD, 3KSZ, 3KV3, 3L4S, 3L6O, 3LC1, 3LC2, 3LC7, 3LVF

  • PubMed Abstract: 

    The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD(+). A new phosphate recognition site, "new P(i)" site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 A away from the "classical P(i)" site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S+H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the "P(s)" site in the ternary complexes but at the "new P(i)" site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the "new P(i)" site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the "flip-flop" model proposed for the enzyme mechanism.


  • Organizational Affiliation

    Department of Biotechnology, Indian Institute of Technology, Kharagpur, Pin-721302, West Bengal, India.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Glyceraldehyde-3-phosphate dehydrogenase 1A [auth Q],
B [auth R],
C [auth O],
D [auth P]
336Staphylococcus aureus subsp. aureus MRSA252Mutation(s): 2 
Gene Names: gapgap1gapAgapA1SAR0828
EC: 1.2.1.12
UniProt
Find proteins for Q6GIL8 (Staphylococcus aureus (strain MRSA252))
Explore Q6GIL8 
Go to UniProtKB:  Q6GIL8
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ6GIL8
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.205 
  • R-Value Observed: 0.208 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 66.654α = 90
b = 94.315β = 106.02
c = 87.981γ = 90
Software Package:
Software NamePurpose
d*TREKdata scaling
MOLREPphasing
REFMACrefinement
PDB_EXTRACTdata extraction
StructureStudiodata collection
d*TREKdata reduction

Structure Validation

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Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted NADClick on this verticalbar to view details

Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-08-18
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Source and taxonomy, Version format compliance
  • Version 1.2: 2013-12-11
    Changes: Database references
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-11-01
    Changes: Data collection, Refinement description