Even though C115 is only modestly important for catalysis, its replacement by serine led to a 117-fold reduction in activity (Table ), perhaps due to the introduction of a detrimental interaction. The activities of R32A-and R56A-PaHisGS decreased 25-and 42fold, respectively, in comparison with the WT-PaHisGS (Table ). This demonstrates the importance of these residues in PaHisGS catalysis, possibly because R56 and R32 may contribute to leaving group departure at the transition state. K57 is adjacent to R56 in the PaHisGS primary sequence, but in all PaHisGS and PaATPPRT crystal structures, it points away from the active site. We hypothesised that in the absence of the R56 guanidinium group, the K57 -NH3 + group could move towards the active site and assist in leaving group departure. However, the R56A/K57A-PaHisGS double mutant displayed a 254-fold decrease in activity (Table ), which is only ~6-fold more catalytically impaired than the R56A-PaHisGS, indicating just a modest catalytic importance for K57. R32, R56, K57, and C115 are highly conserved in HisGS across species, and D179 is also conserved but sometimes replaced with a glutamate residue. Nevertheless, out of these five residues in PaHisGS, only the arginine residues seem to be significantly important for catalysis. . No activity was detected when the PaHisGS mutant-catalysed reactions were carried out in the presence of bovine serum albumin (BSA) (Supplementary Fig. ), ruling out that allosteric rescue was due to nonspecific protein binding. To gather orthogonal evidence for the allosteric rescue, the reaction catalysed by each PaHisGS mutant was analysed by P-NMR spectroscopy in the presence and absence of PaHisZ (Supplementary Fig. ) under conditions where product can be detected with WT-PaHisGS. The characteristic chemical shift at ~3.30 ppm (Fig. )