Abstract

cis-Prenyltransferase (cPT) synthesizes long-chain polyprenols, with product length dictated by its catalytic pocket architecture. This study characterized a novel cPT from the red alga Porphyra umbilicalis (PucPT), which produces C45 and C50 cis-polyprenols from FPP and IPP in vitro and in vivo enzymatic assays. Molecular dynamics and site-directed mutagenesis revealed that pocket volume critically modulates catalytic efficiency. Under identical product chain lengths, the F138A mutation (2691.4 Å3 to 2747.4 Å3) enhanced product yield by 4.5%, whereas the A157F mutation (2691.4 Å3 to 2282.5 Å3) reduced yield by 39.7%. Furthermore, introducing positive charges via E141R and E285R mutations stabilized intermediates, yielding longer chains (C145 and C110, respectively) yet reducing total yields by 27.9% and 42.9%. These findings demonstrate a trade-off between chain length and efficiency, governed by synergistic geometric constraints and electrostatic interactions. This work elucidates the structural determinants of cPT specificity, providing a theoretical foundation for engineering biocatalysts to synthesize tailored cis-polyprenols.