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. 2023 Apr 5;14(5):921-933.
doi: 10.1039/d3md00058c. eCollection 2023 May 25.

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

Rasangi Pathirage  1 , Lorenza Favrot  2 , Cecile Petit  2 , Melvin Yamsek  2 , Sarbjit Singh  3 , Jayapal Reddy Mallareddy  3 , Sandeep Rana  3 , Amarnath Natarajan  1   3   4   5 , Donald R Ronning  1
Affiliations

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

Rasangi Pathirage et al. RSC Med Chem. .

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 ICapp50 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.

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Conflict of interest statement

There are no conflicts to declare. The research presented here was not supported by nor represents experiments performed by BioNTech.

Figures

Fig. 1
Fig. 1. Structure of the pyruvate (PYR) binding site in M. tb citrate synthase. The red sphere represents the oxygen atom of the water molecule (W) interacting with pyruvate. (A) The electron density of a Fo − Fc omit map is shown contoured at 3σ (orange); PYR and W residues were omitted during map calculation. (B) The bonded lengths of interactions formed by pyruvate with the adjacent residues. (C) Alignment of the pyruvate bound CitA crystal structure (light blue) with the ligand free CitA crystal structure (cyan).
Fig. 2
Fig. 2. Allosteric binding site differences between the NADH bound E. coli citrate synthase (light green) and M. tb GltA2 (brownish purple) with pyruvate (PYR) bound M. tb CitA (light blue). Helices of the E. coli citrate synthase are labelled in dark green and helices of M. tb CitA are labelled in purple.
Fig. 3
Fig. 3. Superposition of CitA wild-type (light blue) with R149E (wheat) and R153M (light pink) variants shows only minor backbone variation and minor changes within the pyruvate binding pocket (inset).
Fig. 4
Fig. 4. (A) Michaelis–Menten curves plotted for CitA wild-type (WT) and R149E, R153M variants with the table showing the data obtained from kinetic analysis using experimental results performed in triplicate. (B) The bar graph showing the percentage enzyme activity of CitA wild-type and the variants in the absence or presence of 500 μM pyruvate.
Fig. 5
Fig. 5. (A) The reaction between Ebselen and C143 residue (B) the bar graph showing the percentage activity difference between Ebselen modified CitA (CitA-EBS) versus unmodified wild-type CitA (CitA-WT) and recovery of CitA activity in presence of a reductant (C) the alignment of CitA unmodified ligand free structure (cyan), pyruvate bound CitA structure (light blue) and Ebselen modified CitA structure (gray) and the electron density of composite omit map for Ebselen modification of C143 residue.
Fig. 6
Fig. 6. IC50 curves for Michael acceptor compounds. (A) The IC50 curve for the 28-131 and the reaction between 28-131 and C143. (B) The IC50 curve for the 28-171 and the reaction between 28-171 and C143.
Fig. 7
Fig. 7. The alignment of citrate bound Ebselen modified M. tb CitA structure (grey) and pyruvate bound M. tb CitA structure highlighting the regulatory domain (green) and helices that connect the regulator domain to the active site (orange).
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