All mutants were probed for cHP production using the same activity assay performed on the WT as well as the trapped acyl-enzyme intermediate assay (Supplementary Figure ). Fig. shows that only S26C and Y167F from the active site variants could still produce cHP albeit with a lower yield. This result is akin to the trend in activity published by Bourgeois et al. who also mutated the catalytic Tyr in three different XYP CDPSs which still produced their respective products. This demonstrates that the phenyl ring is vital in substrate binding rather than the hydrogen bonding interactions from the phenolic hydroxyl. 36, When the H-bond between S26 and D58 is disrupted by mutating D58 to either an alanine or an asparagine, the enzyme is inactive. This highlights the essential role that D58 has in potentially polarizing and positioning the serine in the active site for substrate binding and acyl enzyme formation. By mutating the P1 residues, the variants were still capable of accepting histidine with only one mutant -E174H -displaying no activity. E174H was designed to reverse the charge of the residue and was predicted to repel the incoming histidine from P1. Further investigation into the changes imposed by these mutants was performed by a, enhanced image of the WT structure highlighting the two sets of residues targeted for mutagenesis -active site (pink) and pocket 1 (turquoise). b, cHP activity assay for each set of mutants -the activity is shown as a percentage of the wild-type ParcuCDPS activity. Each residue is shown in a different colour and the patterned bar represents a second mutation of the same residue. The overall trend shown is a decrease in the capability of the mutants to produce cHP with S26C displaying the highest yield. c, Quantification of cyclo(Leu-Pro) production using ParcuCDPS variants. Pro-tRNA was used in combination with Leu-tRNA and Leu-DBE to investigate the use of different substrates on product yield. d, quantification of cyclo(Phe-Pro) using the same mutants as tested for cLP. solving the crystal structures of a select few (Supplementary Figure , Supplementary Table ).