Fig. 2: Substrates used to probe mechanism of NdasCDO, effect of pH, temperature and salt concentration in the reaction.

a Criteria for usage of distinct CDP substrates for different experiments: cGG is a reaction control as it cannot be oxidized; catalysis with cFG is slow and a single oxidation is possible – oxidation leads to a small change in absorbance at 297 nm; cFP undergoes fast oxidation, and two oxidations are possible – oxidation leads to a large change in absorbance at 312 nm; (hydrogen atoms depicted purple when participating in the first oxidation and blue when participating in the second oxidation rection); cWS undergoes slow oxidation, and although two oxidations are possible, second is less favourable – oxidation leads to a large change in absorbance at 297 nm; b pH dependence of log (k_cat) and log (k_cat/K_M) for varied cFG (blue and purple, respectively) examined using a mixed buffer system. Data were obtained at 30 °C whilst varying the concentration of substrate. log (k_cat) data are fit to a 1-proton dependence (Eq. (2)), log (k_cat/K_M) are fit to a 2-proton dependence equation (Eq. (3)), shown as mean values ± standard error of the fit across three individual replicates. Errors for k_cat/K_M and k_cat/K_M were propagated appropriately. c Melting curves using differential scanning fluorimetry (DSF), raw data (circles) fitted to a Boltzmann sigmoidal equation (lines); T_m values after fitting are shown with varying NaCl concentrations. d Top, snapshot of mass photometry movie, which was recorded for 120 s. Data was processed using the DiscoverMP software to generate the mass distribution histograms displayed in the bottom. Under these conditions a predominant species of 205 ± 47 kDa is observed. e Temperature dependence of k_cat, data were fitted to Eq. (1) yielding ΔH^‡ = 4.06 ± 2.42 kcal mol⁻¹ and ΔS^‡ of −0.049 ± 0.008 kcal mol⁻¹. Data shown as mean values ± standard error of the fit across three individual replicates.