DSF indicated that the additional mutation does not alter the Tm of the protein (Fig. ) as compared with R56A/K57A-PaHisGS or WT-PaHisGS Tm. Furthermore, PRPP led to an increase in Tm, showing the triple mutant can bind this substrate. As expected, PRATP formation could not be detected with R32A/R56A/K57A-PaHisGS as catalyst (Fig. ).