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  • Linezolid Purification of Rv c In order to

    2024-02-27

    Purification of Rv2477c. In order to investigate the catalytic capabilities of Rv2477c, we expressed the protein as an N-terminal histidine-tagged fusion protein in E. coli BL21 cells and purified the protein by cobalt-affinity chromatography. As shown in Fig. 2A, the 65 kDa fusion protein was expressed strongly in the heterologous host Linezolid and high quantities of Rv2477c protein was eluted from the affinity chromatography column. The pooled elute fractions were subjected to six cycles of ultrafiltration using a 50,000 MWCO membrane filter and concentrated to a volume of 0.6 ml. As shown in Fig. 2B, the resultant protein preparation analyzed by SDS-PAGE and Coomassie staining showed a single band when 4 μg (twice the quantity used in assays) was resolved. A yield of 0.6 mg purified protein was obtained. The ATPase activity of Rv2477c is Mn-dependent and is maximal at acidic pH. Biochemical characterization of Rv2477c was performed using 2 μg (1 μM) of the protein, unless indicated otherwise. The ATPase activity of Rv2477c was stimulated by the presence of divalent cations among which Mn2+ showed the highest stimulation followed by Co2+ (Fig. 2C). Rv2477c exhibited maximal activity at pH 5.2 (Fig. 2D). The hydrolytic activity of purified Rv2477c increased with concentration of ATP approaching saturation around 2000 μM (Fig. 2E). Rv2477c displayed typical Michaelis-Menten kinetics in its ATPase activity. The apparent Vmax was 45.5 ± 1.8 nmol/mg/min and the Km was 90.5 ± 15.3 μM. The kcat was 187.2 h−1. Catalytic efficiency (kcat/Km) was 2069 mM−1 h−1. The ATPase activity of Rv2477c increased linearly with protein concentration (Fig. 2F) and time (Fig. 2G). The Rv2477c protein also showed hydrolase activity against GTP, TTP and CTP but these activities were lower than its ATPase activity (Fig. 2H). The non-hydrolysable analog ATPγS inhibited the ATPase activity of Rv2477c in a dose-dependent manner. Orthovanadate and KNO3 also inhibited the ATPase activity (Table 1). The Walker B glutamate residues are critical for the ATPase activity of Rv2477c. Since the glutamate residues in the Walker B motif have been shown to be vital for ATPase activity in other ATPases [14], [15], we investigated their role in Rv2477c. The Rv2477c-EQ2 mutant protein containing glutamate to glutamine substitutions at positions 185 and 468 in the two nucleotide-binding domains was generated by site-directed mutagenesis and purified (Fig. 3A and B). The ATPase activity of the Rv2477c-EQ2 mutant protein was severely inhibited and was about 10% of the activity in the wild-type Rv2477c protein (Fig. 3C). Thus, these glutamate residues are critical for the ATPase activity of Rv2477c. Tetracycline and erythromycin inhibit the ATPase activity of Rv2477c. Rv2477c exhibits amino acid identities with Vga(A) which has been shown to be an ATPase involved in antibiotic resistance [16], [17]. Furthermore, Rv2477c is homologous to EttA, the ABC-F protein in E. coli that has been shown to associate with the ribosome [8]. It has been reported that the ATPase activity of the ABC-F protein Vga(A) was inhibited by the antibiotic pristinamycin IIA and Vga(A) was capable of displacing antibiotic from the ribosome in vitro[16], [17]. Therefore, we investigated whether antibiotics affected the ATPase activity of Rv2477c. We pre-incubated the purified Rv2477c protein with different concentrations of antibiotics known to bind ribosomal subunits and then assayed the ATPase activity of Rv2477c. We found that tetracycline and erythromycin inhibited the ATPase activity of Rv2477c (Table 2). Kanamycin and streptomycin did not significantly affect the ATPase activity of Rv2477c.
    Discussion Mtb causes severe disease and more than 2 billion persons are latently infected with the pathogen [1]. Latent tuberculosis infection occurs when the pathogen enters a dormant state and persists in the human body without causing active clinical disease [2]. Latently infected people serve as “seedbeds” for active tuberculosis since the pathogen is able to reactivate and cause active tuberculosis when the immune system is suppressed [18]. The pathogen enters a non-replicating state during dormancy as oxygen and nutrient availability become limited due to the immune response. When the conditions become favorable for growth, the pathogen reawakens from its dormant state [2]. Dormancy-associated metabolic pathways in the pathogen are likely to be critical targets for the development of therapies that treat and cure latent tuberculosis infection.