The Rv c EQ protein was purified from ml

The Rv2477c-EQ2 protein was purified from 600 ml induced cultures following the same protocol described above. Elution of the mutant protein from the affinity resin was done using 200 mM imidazole. After concentration and buffer-exchange of protein as described above, the purity of the protein was confirmed as above. ATP hydrolysis assays. The ATPase activity assays were carried out in a 30 μl reaction mix containing 2 μg (1 μM) of purified Rv2477c (or Rv2477c-EQ2) protein, 500 μM ATP, 100 mM sodium acetate AZD0156 5.2, 100 mM NaCl, 2.5 mM MnCl2 and 10% glycerol. ATP (Calbiochem- Millipore, Burlington, MA) was dissolved in 100 mM Tris-HCl pH 7.0 to make a fresh 10 mM stock solution from which appropriate amounts were added to assay. ATP concentrations between 50 and 2000 μM were used to determine the kinetic parameters. A 2 mM excess concentration of divalent cation over ATP concentration was maintained throughout. The apparent maximal reaction velocity (Vmax) and Michaelis-Menten constant (Km) were determined using the Enzyme Kinetics Nonlinear Fit Results from Michaelis-Menten plots in SigmaPlot 13 (Systat Software, Inc., San Jose, CA). In pH-dependence assays, 100 mM sodium acetate pH 4.0 or 100 mM Tris-HCl pH 7.0/7.5/8.0/8.8 were substituted appropriately. In assays for divalent cation dependence, 2.5 mM MgCl2 or CaCl2 or CoCl2 or ZnSO4 were used instead of MnCl2. GTP (MP Biomedicals, Solon, OH), CTP (Alfa Aesar, Ward Hill, MA) or TTP (Sigma Aldrich, St. Louis, MO) were substituted for ATP in appropriate assays. The malachite green reagent (MGR) assay was used to determine the amount of phosphate released by the ATP hydrolase activity of purified Rv2477c following a previously reported procedure [12]. MGR is a 2:1:1:2 mixture of 0.0812% (w/v) malachite green, 2.32% (w/v) polyvinyl alcohol, 5.72% (w/v) ammonium molybdate in 6 M HCl and water, respectively. After incubation at 37 °C for 30 min, 150 μl MGR and 15 μl of 34% (w/v) sodium citrate were added. After 30 min at 22 °C, absorbance at 620 nm was measured using an accuSkan™ GO UV/Vis Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Absorbance readings of negative control reactions without Rv2477c protein were subtracted from respective readings in reactions containing the protein. The quantity of inorganic phosphate released was estimated using a standard curve that was linear up to 200 μM in the reaction buffer. The specific activity of the purified protein was determined from the rate of inorganic phosphate released from ATP. The effects of ATPγS (TOCRIS Bioscience, Bristol, UK) which is the non-hydrolysable substrate analog and known inhibitors of ATPases (sodium orthovanadate, sodium azide and KNO3) on ATPase activity were investigated by pre-incubating the purified Rv2477c protein with the substrate analog or inhibitor for 15 min on ice prior to assay. Sodium orthovanadate (MP Biomedicals, Solon, OH) was prepared by following a previously described protocol [13]. To investigate the effects of antibiotics on the ATPase activity of Rv2477c, the purified protein was pre-incubated for 15 min at room temperature with antibiotic prior to addition of ATP. Antibiotic stock solutions were prepared in water with the exception of erythromycin which was prepared in ethanol. Ethanol solvent control was used for erythromycin in assays.
Results The mycobacterial protein Rv2477c shows similarities with bacterial ABC-F subfamily proteins involved in ribosome function and antibiotic resistance. The protein encoded by the Rv2477c gene is an essential gene annotated in the mycobacterial genome as a “probable macrolide transport ATP-binding protein/ABC transporter” [11]. The Rv2477c protein does not contain any predicted transmembrane domains and displays homology to the ABC-F protein EttA in E. coli that controls ribosomal processes [8]. The ABC-F subfamily members are soluble proteins that lack transmembrane domains but contain tandem nucleotide-binding domains and are involved in diverse cellular processes such as translation, cell division and antibiotic resistance [5]. Several genes that encode lipid metabolizing enzymes are located in the genomic neighborhood of Rv2477c. A similar genetic organization is seen in other mycobacteria (Fig. 1A). The ABC signature motifs and the canonical Walker A and Walker B motifs were conserved on Rv2477c (Fig. 1B).