was kept protonated in line with the mechanism shown in Figure . All other residues (except H50 in PriA, which was doubly protonated) were kept in their default protonation states at physiological pH determined by use of PROPKA 3.1, 60 and visual inspection. The substrates ProFAR and PRA were manually placed into the relevant active sites in the same conformation as found in the structure of the HisA wild-type enzyme in complex with ProFAR and in the case of PRA (Figure ), was placed by manual overlay of the reactive part of PRA with the reactive part of ProFAR, and with the carboxylate group of PRA keeping key interactions with active site residues. Partial charges for ligands ProFAR, PRA, PRFAR and CdRP, were calculated using the standard restrained electrostatic potential (RESP) protocol using Antechamber v. 17.3, 61 and based on the vacuum electrostatic potential calculated at the HF/6-31G(d) level of theory, using Gaussian 09