8GMK

Pyruvate bound structure of Citrate Synthase (CitA) in Mycobacterium Tuberculosis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.81 Å
  • R-Value Free: 0.260 
  • R-Value Work: 0.210 
  • R-Value Observed: 0.212 

Starting Model: experimental
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Ligand Structure Quality Assessment 


This is version 2.0 of the entry. See complete history


Literature

Mycobacterium tuberculosis CitA activity is modulated by cysteine oxidation and pyruvate binding.

Pathirage, R.Favrot, L.Petit, C.Yamsek, M.Singh, S.Mallareddy, J.R.Rana, S.Natarajan, A.Ronning, D.R.

(2023) RSC Med Chem 14: 921-933

  • DOI: https://doi.org/10.1039/d3md00058c

  • PubMed Abstract: 

    As an adaptation for survival during infection, Mycobacterium tuberculosis becomes dormant, reducing its metabolism and growth. Two types of citrate synthases have been identified in Mycobacterium tuberculosis , GltA2 and CitA. Previous work shows that overexpression of CitA, the secondary citrate synthase, stimulates the growth of Mycobacterium tuberculosis under hypoxic conditions without showing accumulation of triacylglycerols and makes mycobacteria more sensitive to antibiotics, suggesting that CitA may play a role as a metabolic switch during infection and may be an interesting TB drug target. To assess the druggability and possible mechanisms of targeting CitA with small-molecule compounds, the CitA crystal structure was solved to 2.1 Å by X-ray crystallography. The solved structure shows that CitA lacks an NADH binding site that would afford allosteric regulation, which is atypical of most citrate synthases. However, a pyruvate molecule is observed within the analogous domain, suggesting pyruvate may instead be the allosteric regulator for CitA. The R149 and R153 residues forming the charged portion of the pyruvate binding pocket were mutated to glutamate and methionine, respectively, to assess the effect of mutations on activity. Protein thermal shift assay shows thermal stabilization of CitA in the presence of pyruvate compared to the two CitA variants designed to decrease pyruvate affinity. Solved crystal structures of both variants show no significant structural changes. However, the catalytic efficiency of the R153M variant increases by 2.6-fold. Additionally, we show that covalent modification of C143 of CitA by Ebselen completely arrests enzyme activity. Similar inhibition is observed using two spirocyclic Michael acceptor containing compounds, which inhibit CitA with IC app 50 values of 6.6 and 10.9 μM. A crystal structure of CitA modified by Ebselen was solved, but significant structural changes were lacking. Considering that covalent modification of C143 inactivates CitA and the proximity of C143 to the pyruvate binding site, this suggests that structural and/or chemical changes in this sub-domain are responsible for regulating CitA enzymatic activity.


  • Organizational Affiliation

    Department of Pharmaceutical Sciences, University of Nebraska Medical Center Omaha NE 68198 USA [email protected].


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
citrate synthase
A, B
379Mycobacterium tuberculosis H37RvMutation(s): 0 
Gene Names: citASAMEA2683035_02214
EC: 2.3.3.16
UniProt
Find proteins for P9WPD3 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P9WPD3 
Go to UniProtKB:  P9WPD3
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP9WPD3
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
PGE
Query on PGE

Download Ideal Coordinates CCD File 
D [auth A]TRIETHYLENE GLYCOL
C6 H14 O4
ZIBGPFATKBEMQZ-UHFFFAOYSA-N
PEG
Query on PEG

Download Ideal Coordinates CCD File 
AA [auth B],
J [auth A],
L [auth A],
N [auth A],
X [auth B]
DI(HYDROXYETHYL)ETHER
C4 H10 O3
MTHSVFCYNBDYFN-UHFFFAOYSA-N
PYR (Subject of Investigation/LOI)
Query on PYR

Download Ideal Coordinates CCD File 
C [auth A],
Q [auth B]
PYRUVIC ACID
C3 H4 O3
LCTONWCANYUPML-UHFFFAOYSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
E [auth A]
F [auth A]
G [auth A]
H [auth A]
I [auth A]
E [auth A],
F [auth A],
G [auth A],
H [auth A],
I [auth A],
K [auth A],
M [auth A],
O [auth A],
P [auth B],
R [auth B],
S [auth B],
T [auth B],
U [auth B],
V [auth B],
W [auth B],
Y [auth B],
Z [auth B]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.81 Å
  • R-Value Free: 0.260 
  • R-Value Work: 0.210 
  • R-Value Observed: 0.212 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 99.524α = 90
b = 99.524β = 90
c = 141.345γ = 120
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data scaling
HKL-2000data reduction
PHENIXphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)United StatesCA260749

Revision History  (Full details and data files)

  • Version 1.0: 2023-06-07
    Type: Initial release
  • Version 1.1: 2023-11-08
    Changes: Data collection, Source and taxonomy
  • Version 2.0: 2023-11-15
    Changes: Atomic model, Data collection