Following on from these results, we designed Generation 2 of double mutants from P1 residues (variants Y55F+Y189F, Y55F+E174A, Y189F+E174A, E174L+Y189L), aiming to severely perturb the activity of the enzyme towards histidine. When we investigated the formation of cHP using Generation 2 variants, all mutants synthesised significantly less cHP than the single Gen 1 mutants alone (Fig. ). This suggests that binding to the P1 pocket is not facilitated by a single residue alone, rather by a combination of interactions. The impact of mutating Y55 carries more weight than Y189, suggesting it may directly interact with the histidine residue via a H-bond rather than adding to the polar surface. A similar pattern of residues interacting with the imidazole from histidine is seen in HisRS, in which two tyrosine and a glutamate residue mediate interactions with the nitrogen groups of histidine (Supplementary Figure ).