4DEF

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.64 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.189 
  • R-Value Observed: 0.190 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis.

Pegan, S.D.Rukseree, K.Capodagli, G.C.Baker, E.A.Krasnykh, O.Franzblau, S.G.Mesecar, A.D.

(2013) Biochemistry 52: 912-925

  • DOI: https://doi.org/10.1021/bi300928u
  • Primary Citation of Related Structures:  
    4DEF, 4DEL

  • PubMed Abstract: 

    Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.


  • Organizational Affiliation

    Eleanor Roosevelt Institute and Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States. spegan@du.edu


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Fructose-bisphosphate aldolase349Mycobacterium tuberculosisMutation(s): 0 
Gene Names: fbaMT0379MTCY13E10.25cRv0363c
EC: 4.1.2.13
UniProt
Find proteins for P9WQA3 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P9WQA3 
Go to UniProtKB:  P9WQA3
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP9WQA3
Sequence Annotations
Expand
  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.64 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.189 
  • R-Value Observed: 0.190 
  • Space Group: I 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 60.655α = 90
b = 119.708β = 90
c = 164.826γ = 90
Software Package:
Software NamePurpose
PHASERphasing
REFMACrefinement
PDB_EXTRACTdata extraction
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-01-23
    Type: Initial release
  • Version 1.1: 2013-06-26
    Changes: Database references
  • Version 1.2: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description