Electrostatic interaction and regioselectivity enhancement in proline cis-4-hydroxylase for L-pipecolic acid hydroxylation

L-Proline cis-4-hydroxylase (cis-P4H), a non-heme Fe²⁺/α-ketoglutarate-dependent dioxygenase (KDD), finds application in proline C4 hydroxylation and selective L-pipecolic acid (L-PA) C5 hydroxylation. Nonetheless, its regioselectivity is often ambiguous, yielding nearly equal amounts of cis-5/cis-3 hydroxylated L-PA isomers, posing challenges in separation and purification. In this study, we selected Kordia jejudonensis proline hydroxylase (KjPH) for modification and investigated the electrostatic effect's molecular mechanism on its regioselectivity. Through sequence and catalytic domain alignment of KjPH (cis-5/cis-3 = 20:1), SmP4H (cis-5/cis-3 = 1:1, from Sinorhizobium meliloti), and MlP4H (cis-5/cis-3 = 1:7, from Mesorhizobium loti), we identified four non-conserved key residues (Y35, S57, F95, and C97). We confirmed that F95 in KjPH plays a pivotal role in affecting regioselectivity. The single-site variant F95Y significantly enhanced regioselectivity, increasing the cis-5/cis-3 ratio from 20:1 to 55:1. Molecular dynamics simulations unveiled that the improved regioselectivity of the F95Y variant primarily resulted from the electrostatic repulsive interaction, which increased the distance between the substrate's C3 site and the Fe²⁺ catalytic core.