Addressing the Evolution of Cardenolide Formation in Iridoid-Synthesizing Plants: Site-Directed Mutagenesis of PRISEs (Progesterone-5β-Reductase/Iridoid Synthase-like Enzymes) of Plantago Species

Enzymes capable of processing a variety of compounds enable plants to adapt to diverse environmental conditions. PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), examples of such substrate-promiscuous enzymes, are involved in iridoid and cardenolide pathways and demonstrate notable substrate promiscuity by reducing the activated C=C double bonds of plant-borne and exogenous 1,4-enones. In this study, we identified PRISE genes in <i>Plantago media</i> (<i>PmdP5βR1</i>) and <i>Plantago lanceolata</i> (<i>PlP5βR1</i>), and the corresponding enzymes were determined to share a sequence identity of 95%. Despite the high sequence identity, recombinant expressed <i>Pmd</i>P5βR1 was 70 times more efficient than <i>Pl</i>P5βR1 for converting progesterone. In order to investigate the underlying reasons for this significant discrepancy, we focused on specific residues located near the substrate-binding pocket and adjacent to the conserved phenylalanine "clamp". This clamp describes two phenylalanines influencing substrate preferences by facilitating the binding of smaller substrates, such as 2-cyclohexen-1-one, while hindering larger ones, such as progesterone. Using structural analysis based on templates PDB ID: 5MLH and 6GSD from PRISE of <i>Plantago major</i>, along with in silico docking, we identified positions 156 and 346 as hot spots. In <i>Pl</i>P5βR1 amino acid residues, A156 and F346 seem to be responsible for the diminished ability to reduce progesterone. Moreover, the double mutant <i>Pl</i>P5βR_F156L_A346L, which contains the corresponding amino acids from <i>Pmd</i>P5βR1, showed a 15-fold increase in progesterone 5β-reduction. Notably, this modification did not significantly alter the enzyme's ability to convert other substrates, such as 8-oxogeranial, 2-cyclohexen-1-one, and methyl vinyl ketone. Hence, a rational enzyme design by reducing the number of hotspots selectively, specifically improved the substrate preference of <i>Pl</i>P5βR1 for progesterone.