Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase.

Itaconate is a mammalian antimicrobial metabolite that inhibits the isocitrate lyases (ICLs) of Mycobacterium tuberculosis. Herein, we report that ICLs form a covalent adduct with itaconate through their catalytic cysteine residue. These results reveal atomic details of itaconate inhibition and provide insights into the catalytic mechanism of ICLs.

Acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2.

Isocitrate lyase is important for lipid utilisation by Mycobacterium tuberculosis but its ICL2 isoform is poorly understood. Here we report that binding of the lipid metabolites acetyl-CoA or propionyl-CoA to ICL2 induces a striking structural rearrangement, substantially increasing isocitrate lyase and methylisocitrate lyase activities. Thus, ICL2 plays a pivotal role regulating carbon flux between the tricarboxylic acid (TCA) cycle, glyoxylate shunt and methylcitrate cycle at high lipid concentrations, a mechanism essential for bacterial growth and virulence.

Targeting isocitrate lyase for the treatment of latent tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that can remain dormant for many years before becoming active. One way to control and eliminate TB is the identification and treatment of latent TB, preventing infected individuals from developing active TB and thus eliminating the subsequent spread of the disease. Isocitrate lyase (ICL) is involved in the mycobacterial glyoxylate and methylisocitrate cycles. ICL is important for the growth and survival of M. tuberculosis during latent infection. ICL is not present in humans and is therefore a potential therapeutic target for the development of anti-TB agents. Here, we explore the evidence linking ICL to persistent survival of M. tuberculosis. The structure, mechanism and inhibition of the enzyme is also discussed.

Development of NMR and thermal shift assays for the evaluation of Mycobacterium tuberculosis isocitrate lyase inhibitors.

The enzymes isocitrate lyase (ICL) isoforms 1 and 2 are essential for Mycobacterium tuberculosis survival within macrophages during latent tuberculosis (TB). As such, ICLs are attractive therapeutic targets for the treatment of tuberculosis. However, there are few biophysical assays that are available for accurate kinetic and inhibition studies of ICL in vitro. Herein we report the development of a combined NMR spectroscopy and thermal shift assay to study ICL inhibitors for both screening and inhibition constant (IC50) measurement. Operating this new assay in tandem with virtual high-throughput screening has led to the discovery of several new ICL1 inhibitors.