An in vitro system to study cyclopeptide heterophyllin B biosynthesis in the medicinal plant Pseudostellaria heterophylla

Abstract

Plant cyclopeptides are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. Many of the cyclopeptides have highly unusual structures and potent biological activities. However, the majority of the cyclopeptides have not been studied for their biosynthetic mechanisms. In this study, we have established a culture system for the biosynthetic study of heterophyllin B (HB), a cyclopeptide produced by the medicinal plant Pseudostellaria heterophylla. We first developed a shoot culture of P. heterophylla that produced HB consistently under laboratory conditions. Using 14C-labeled proline as tracer, we showed that labeled HB was produced by the cultured shoots, indicating that this system has de novo biosynthetic activity. Next, we chemically synthesized HB’s linear peptide precursor (LHB) and the N-acetyl cysteamine thioester of LHB (LHB-SNAC). When LHB-SNAC was incubated with total cell free extracts of the cultured shoots, a small amount of cyclized product (HB), in addition to the hydrolyzed product (LHB), was produced. The in vivo and in vitro results demonstrate the presence of an HB biosynthetic system, which provides insight into the molecular mechanism for plant cyclopeptide biosynthesis.