Figure 2 | Scientific Reports

Figure 2

From: Identification of small molecules targeting homoserine acetyl transferase from Mycobacterium tuberculosis and Staphylococcus aureus

Figure 2

Biochemical characterization of Rv3341. (A) Oligomeric state of Rv3341 enzyme. Analytical ultracentrifugation was performed using purified (His)6-MetX at 10 µM, 20 µM and 30 µM concentration in 20 mM Tris–Cl buffer, pH-7.5 containing 150 mM NaCl. (B) Enzymatic activity of different oligomeric forms of Rv3341. The amount of CoA released in enzymatic reactions in the presence of 0.5 µM of monomer, dimer or tetramer form of Rv3341 was determined. (C) Michaelis Menten plot for Rv3341 enzymatic activity. The enzymatic reactions were performed in vitro using 1 mM L-homoserine and different concentrations of acetyl-CoA. (D,E) Substrate specificity of Rv3341 enzyme. In order to determine the substrate specificity of Rv3341 for acetyl-CoA (D), enzymatic reactions were performed in the presence of either 1.0 mM acetyl-CoA (AC) or propionyl-CoA (PC) or succinyl-CoA (SC). The specificity for L-homoserine was determined by performing enzymatic reaction in the presence of either 1.0 mM L-homoserine (L-HS) or L-serine (L-Ser) or L-threonine (L-Thr). (F) Effect of buffer pH on Rv3341 activity in vitro. HSAT activity assays were performed in buffers of pH in the range of 6.0–9.0. (G) Time kinetics analysis of Rv3341 activity. The HSAT activity was calculated after 10 min, 20 min, 40 min and 60 min post-incubation with substrates. (H) HSAT activity of wild type and mutant proteins. The enzymatic assays were performed using 0.5 µM of purified wild type and mutant protein in assay conditions containing 1.0 mM L-homoserine and 1.0 mM acetyl-CoA. (I) Inhibition of Rv3341 activity by SAM. Rv3341 enzymatic activity was calculated in presence of 100 µM of SAM. The amount of coenzyme A released in the enzymatic reactions was determined upon the addition of Ellman reagent and measuring absorbance at 412 nm. The data shown on y-axis in panels B-H is µM CoA released in enzymatic reaction and values shown are mean ± S.E. obtained from three independent experiments performed in duplicates. The data shown in panel I is mean ± S.E. of percentage inhibition obtained from two independent experiments performed in duplicates.

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