C115S-, R32A-, R56A-, and R56A/K57A-PaHisGS are catalytically compromised. No PRATP formation could be detected above the background noise of the assay (no-enzyme control) during the initial activity screen when either C115S-, R32A-, R56A-, or R56A/K57A-PaHisGS was used as catalyst (Fig. ), indicating these mutants have impaired catalytic activity. DSF-based thermal denaturation assays showed these mutants display similar thermal unfolding profiles to the WT protein (Fig. ), demonstrating the mutations do not thermally destabilise the tertiary structure of the protein, and data fit to equation (2) yielded melting temperatures (Tm) shown in Supplementary Table . Moreover, as described previously and repeated here for WT-PaHisGS, the presence of PRPP increased the Tm of the mutants (Fig. ; Supplementary Table ), indicating the catalytically impaired PaHisGS variants can bind PRPP, in agreement with the ordered kinetic mechanism proposed for this enzyme. Analytical size-exclusion chromatography produced similar elution profiles for WT-, C115S-, R32A-, R56A-, and R56A/K57A-PaHisGS (Supplementary Fig. ), which includes the expected dimer and a higher oligomeric state. An activity screen with longer reaction times and twice as much enzyme as in the initial screen, to allow more product to accumulate, demonstrated the catalytic ability of C115S-, R32A-, R56A-, and R56A/K57A-PaHisGS is significantly diminished but not abolished (Fig. ). ppm corresponds to the phosphorus in the N 1 -5-phospho-β-D-ribose moiety of PRATP. e Substrate saturation curves for WT and mutant PaATPPRT. Data are mean  standard error of two independent measurements. Lines are best fit of the data to equation (1). f Effect of 1 mM histidine on mutant PaATPPRT-catalysed reaction. Two independent measurements were carried out, and all data points are shown.