Figure 7. In vitro ATPase activity assay for RsBluE.

A. SDS-PAGE-gel of purified, sarkosyl-solubilized RsBluE and SePduX. B. ATPase activity assay performed with ADP-Glo™ ATPase Assay kit (Promega). Reaction mixtures contained HEPES buffer (50 mM, pH 7.0 at 25°C), MgCl₂ (1 mM), ATP (0.1 mM), L-Thr or L-Ser (10 mM), enrichment samples of SePduX, RsBluE, and RsBluE^G99A sarkosyl-solubilized protein (12 μM) incubated at 25°C for 1 h. Negative control reaction mixtures contained extracts of sarkosyl-solubilized protein obtained from cells expressing the empty overexpression vector pTEV5 (vector). ATP to ADP conversion was quantified from the luminescence (relative light units; RLU) after subtracting the background from the no-enzyme control and comparing the value to a standard curve (Fig. S3). Graph titles indicate the growth medium and the substrate constituents of the reaction mixtures used to supplement the growth medium. The source of the protein extracts used in the reactions are in parentheses in bold typeface next to the strain genotype (RsBluE reaction or vector resection). Representative graphs of two independent experiments performed in triplicate. Error bars represent the standard deviation from the mean. C and D. Growth analysis of S. enterica cells grown normoxically at 37°C in NCE minimal medium with glycerol (22 mM), MgSO₄ (1 mM), Cby (1 nM), and filter sterilized RsBluE reaction mixtures (6% v/v) described above (reactions from panel B) containing either ATP (0.08 mM) and L-Thr (0.8 mM, panel C) or L-Ser (0.8 mM, panel D). The final concentrations of substrates after dilution of the filtered reaction mixtures into the medium are in parentheses. Graphs are representative of two independent experiments performed in triplicate. Error bars represent the standard error of the mean. Figure key: Panel C, D: (▲) pduX⁺ strain supplemented with filter sterilized negative control enzymatic reaction containing ATP, and L-Thr (panel C) or L-Ser (panel D) and sarkosyl-solubilized protein extracts from cells carrying the empty vector; (◆) ΔpduX strain supplemented with filter sterilized negative control enzymatic reaction containing ATP, and L-Thr (panel C) or L-Ser (panel D) and sarkosyl-solubilized protein extracts from cells carrying the empty vector; (○) ΔpduX strain supplemented with filtered enzymatic reaction containing ATP, and L-Thr (panel C) or L-Ser (panel D) and purified sarkosyl-solubilized RsBluE protein; (■) ΔcobD strain supplemented with filtered enzymatic reaction containing ATP and L-Thr (panel C) or L-Ser (panel D) and purified sarkosyl-solubilized RsBluE protein.