Fig. 2: Biochemical characterisation of full-length CdaA and comparison with CdaA-DAC.

Activity assays were performed using 1 μM and 16 μM (monomer concentration) of CdaA and CdaA-DAC, respectively; k_cat values were calculated on the assumption that the active enzyme is tetrameric). Buffer was composed of 50 mM HEPES-NaOH pH 8.0, 1 mM MnCl₂ plus 200 mM KCl in a final reaction volume of 0.2 or 0.3 mL. Bars, lines and axes coloured red and blue correspond to full-length CdaA and CdaA-DAC, respectively. All error bars represent the standard error of the mean (SEM) for ≥3 biologically independent replicates. A ATP titration fitted with the Hill equation for CdaA. n = ≥3 biologically independent samples. B ATP titration fitted with the Hill equation for CdaA-DAC. n = ≥3 biologically independent samples. C Testing metal ion-dependent activity of CdaA and CdaA-DAC. Mn²⁺, Co²⁺, Mg²⁺, Ni²⁺ and Cu²⁺ were added to a final concentration of 1 mM. A negative control was performed using the same conditions but without the addition of any metal (not shown). For CdaA n = 3 and for CdaA-DAC n = 2 biologically independent samples. D pH-dependent activity of CdaA and CdaA-DAC. Buffer was composed of 25 mM MES-NaOH, 25 mM HEPES-NaOH, 1 mM MnCl₂ plus 200 mM KCl set to the appropriate pH using 4 N NaOH. pHs above 8.0 were not tested due to formation of metal-based precipitates. For CdaA n = 3 and for CdaA-DAC n = 2 biologically independent samples. E Testing activity of CdaA dependent on the concentrations of ATP and divalent metal ion in buffer composed of composed of 50 mM HEPES-NaOH pH 8.0, 1 mM MnCl₂, 200 mM KCl plus the indicated concentrations of MgCl₂ and ATP. Experiments were also repeated at pH 6.5 with the same result. n = 3 biologically independent samples. F GlmM-dependent inhibition of CdaA and CdaA-DAC. 32 µM GlmM was added to 16 µM CdaA-DAC. 20 µM GlmM was added to 1 µM CdaA. For CdaA n = 3 and for CdaA-DAC n = 3 biologically independent samples.