We used PROPKA to calculate theoretical pKa values for residues in our model at pH 7.0 and generate electrostatic potential maps for each protein variant (calculated data found in Supplementary Table ). From these calculations, P1 is predicted to be mostly neutral apart from Tyr55 and Glu174 which form a negatively charged microenvironment, while P2 is predicted to be mostly lined by positively charged residues. The more positively charged section of P2 could be facilitating the production of cHE -which is also a product of ParcuCDPS (Supplementary Figure ). Glutamate is a large flexible amino acid, likely negatively charged at reaction pH. Thus participation in electrostatic interactions of the Glu tRNAGlu substrate with positively charged residues in P2 is plausible, while specific interactions with Pro tRNAPro are less obvious.