Binding of the Mycobacterium tuberculosis EccCb1 ATPase double hexameric ring to the EsxAB virulence factor is enhanced by ATP.

The EccC enzyme of Mycobacterium tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to the EsxAB factor at the C-terminal region of the D3 domain. In the current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed ∼3.7-fold lower catalytic efficiency than D2 domain and ∼1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd ∼ 11.3 ± 0.6 nM and its affinity is enhanced ∼2 fold in presence of ATP + Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP + Mg2+, and EsxAB + ATP + Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP + Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of antivirulence drugs against M. tuberculosis.

Structural and ATPase activity analysis of nucleotide binding domain of Rv3870 enzyme of M. tuberculosis ESX-1 system.

The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442-747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, Km ~ 100 ± 40 µM, kcat ~ 1.81 ± 0.27 min-1 and Vmax ~ 54.41 µM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70-91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with KD ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high TM ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg2+, as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis.

Structural studies on Mycobacterium tuberculosis HddA enzyme using small angle X-ray scattering and dynamics simulation techniques.

Mycobacterium tuberculosis HddA enzyme phosphorylates the M7P substrate and converts it to M7PP product in GDP-D-α-D-heptose biosynthetic pathway. For structural and functional studies on MtbHddA, we have purified the enzyme, which eluted as a monomer from size exclusion column. Purified MtbHddA had ATPase activity. The SAXS analysis supported globular monomeric scattering profile of MtbHddA in solution. The CD analysis showed that MtbHddA contains 45% α-helix, 18% ß-stands, and 32% random coil structures and showed unfolding temperature (TM) ~ 47.5 °C. The unfolding temperature of MtbHddA is enhanced by 1.78±0.41 °C in ATP+Mg2+ bound state, 2.12±0.41 °C in Mannose bound state and 3.07±0.41 °C in Mannose+ ATP+Mg2+ bound state. The apo and M7P +ATP + Mg2+ complexed models of MtbHddA showed that enzyme adopts a classical GHMP sugar kinase fold with conserved ATP+Mg2+ and M7P binding sites. The dynamics simulation analysis on four MtbHddA models showed that ATP+Mg2+ and M7P binding enhanced the stability of active site conformation of MtbHddA. Our study provides important insights into MtbHddA structure and activity, which can be targeted for therapeutic development against M. tuberculosis.

High fat diet aggravates arsenic induced oxidative stress in rat heart and liver.

Arsenic is a well known global groundwater contaminant. Exposure of human body to arsenic causes various hazardous effects via oxidative stress. Nutrition is an important susceptible factor which can affect arsenic toxicity by several plausible mechanisms. Development of modern civilization led to alteration in the lifestyle as well as food habits of the people both in urban and rural areas which led to increased use of junk food containing high level of fat. The present study was aimed at investigating the effect of high fat diet on heart and liver tissues of rats when they were co-treated with arsenic. This study was established by elucidating heart weight to body weight ratio as well as analysis of the various functional markers, oxidative stress biomarkers and also the activity of the antioxidant enzymes. Histological analysis confirmed the biochemical investigations. From this study it can be concluded that high fat diet increased arsenic induced oxidative stress.