3NBK

Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis in complex with 4'-phosphopantetheine


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.58 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.183 
  • R-Value Observed: 0.184 

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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Kinetic, thermodynamic, and structural insight into the mechanism of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis.

Wubben, T.J.Mesecar, A.D.

(2010) J Mol Biol 404: 202-219

  • DOI: https://doi.org/10.1016/j.jmb.2010.09.002
  • Primary Citation of Related Structures:  
    3NBA, 3NBK

  • PubMed Abstract: 

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine (PhP) to form dephosphocoenzyme A. This reaction sits at the branch point between the de novo pathway and the salvage pathway, and has been shown to be a rate-limiting step in the biosynthesis of CoA. Importantly, bacterial and mammalian PPATs share little sequence homology, making the enzyme a potential target for antibiotic development. A series of steady-state kinetic, product inhibition, and direct binding studies with Mycobacterium tuberculosis PPAT (MtPPAT) was conducted and suggests that the enzyme utilizes a nonrapid-equilibrium random bi-bi mechanism. The kinetic response of MtPPAT to the binding of ATP was observed to be sigmoidal under fixed PhP concentrations, but substrate inhibition was observed at high PhP concentrations under subsaturating ATP concentrations, suggesting a preferred pathway to ternary complex formation. Negative cooperativity in the kinetic response of MtPPAT to PhP binding was observed under certain conditions and confirmed thermodynamically by isothermal titration calorimetry, suggesting the formation of an asymmetric quaternary structure during sequential ligation of substrates. Asymmetry in binding was also observed in isothermal titration calorimetry experiments with dephosphocoenzyme A and CoA. X-ray structures of MtPPAT in complex with PhP and the nonhydrolyzable ATP analogue adenosine-5'-[(α,β)-methyleno]triphosphate were solved to 1.57 Å and 2.68 Å, respectively. These crystal structures reveal small conformational changes in enzyme structure upon ligand binding, which may play a role in the nonrapid-equilibrium mechanism. We suggest that the proposed kinetic mechanism and asymmetric character in MtPPAT ligand binding may provide a means of reaction and pathway regulation in addition to that of the previously determined CoA feedback.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Phosphopantetheine adenylyltransferase
A, B, C, D
177Mycobacterium tuberculosisMutation(s): 0 
Gene Names: coaDkdtBMT3043MTCY349.22Rv2965cu0002e
EC: 2.7.7.3
UniProt
Find proteins for P9WPA5 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P9WPA5 
Go to UniProtKB:  P9WPA5
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP9WPA5
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.58 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.183 
  • R-Value Observed: 0.184 
  • Space Group: P 3 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 114.627α = 90
b = 114.627β = 90
c = 135.206γ = 120
Software Package:
Software NamePurpose
HKL-2000data collection
PHASERphasing
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-09-15
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2023-09-06
    Changes: Data collection, Database references, Derived calculations, Refinement description