Identification and functional characterization of prolyl oligopeptidase involved in the biosynthesis of heterophyllin B in Pseudostellaria heterophylla

As a popular medicinal plant, Pseudostellaria heterophylla has been attached to great importance in herbal medicine. Heterophyllin B (HB), isolated from P. heterophylla, is a strongly pharmacologically active cyclic peptide. However, the key enzymes that catalyze the HB biosynthesis remain unidentified. In this study, we firstly screened a prolyl oligopeptidase (PhPOP1)-coding sequences through transcriptome and gene expression profile analysis, which encodes a prolyl oligopeptidase gene essential for HB biosynthesis. PhPOP1 is highly expressed in P. heterophylla tuberous root phloem and xylem tissues, and localized in the endoplasmic reticulum. Heterologous transient expression assays indicated that PhPOP1 was capable of catalyzing the cyclization of the linear precursor peptide prePhHB into HB. Furthermore, overexpression of PhPOP1 in P. heterophylla root cultures significantly enhanced HB production. Homologous modeling, molecular docking, and site-directed mutagenesis analysis were also conducted and it is discovered that S136, E139, A255, R369, R405, N496, D586, H604 and R664 in the binding pocket regions are vital for the activity of PhPOP1. Taken together, these findings demonstrate that PhPOP1 plays a vital role in HB biosynthesis and therefore is a valuable candidate for increasing HB production in P. heterophylla. The study is the first to conduct a frontier exploration of the HB biosynthesis in P. heterophylla and will provide new insights into the biosynthesis of plant cyclic peptides.