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 14 C-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.

The Biosynthesis of Heterophyllin B in Pseudostellaria heterophylla From prePhHB-Encoded Precursor

“…Most interestingly, prePhHB contained a core peptide (IFGGLPPP) motif. However, this is different from previous reports that the core peptide of HB may be GGLPPPIF (Xu et al, 2012). In this study, we identified, cloned, and characterized the prePhHB from the tuberous root of P. heterophylla via RNA-seq database and the RACE method.…”

“…In addition, CP compounds can form tubular structures and act as membrane ion transferring carriers (Rodriguez-Vazquez et al, 2014). Plant CPs are a large group of small molecule metabolites, typically with 2–37 protein and non-protein amino acids (mainly L-amino acids), which are formed mainly by the peptidic bonds and isolated from the stem barks, leaves, seeds, and roots of a wide variety of higher plants (Condie et al, 2011; Xu et al, 2012). So far, more than 450 plant CPs have been discovered from 26 families of higher plants; they are most commonly found in the Caryophyllaceae , Rhamnaceae , and Violaceae families (Tan and Zhou, 2006; Burman et al, 2010; Pandey et al, 2012).…”

“…Segetalins, belonging to Caryophyllaceae -like CPs, have been demonstrated to be formed by ribosome-dependent linear peptides (Condie et al, 2011). It has also been reported that a small amount of cyclized product (HB) was produced when linear peptide NH2-Gly 1 -Gly 2 -Leu-Pro-Pro-Pro-Ile-Phe-COOH of HB was incubated with crude enzyme extracted from P. heterophylla (Jia et al, 2006; Xu et al, 2012). In addition, plant genomes do not encode nonribosomal peptide synthetases (NRPSs) (Kersten and Weng, 2018).…”

Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

“…SNAC mimics the enzyme-phosphopantetheine moiety that carries enzymatic intermediates in the cyclization enzyme reaction. However, non-enzymatic self-cyclization and non-reactiveness in SNAC derivatives have also been reported 52 53 . We synthesized N -acetoacetyl- L -Ile-SNAC as an intermediate of TeA biosynthesis to investigate the cyclization capability of the KS domain in TAS1; however, synthesized N -acetoacetyl- L -Ile-SNAC showed self-cyclization, the rate of which increased with increasing pH.…”

Antitumor Cyclic Hexapeptides from Rubia Plants: History, Chemistry, and Mechanism (2005–2011)

“…LPS, IFN-γ-induced RAW 264.7 macrophages and HEK-293-NF-κB luciferase stable cell lines were used to examine the effects of RAs on NO production and NF-κB signal pathway. RA-V (17) showed the best activities on NO production inhibition (IC 50 = 0.05 μM) and NF-κB signal pathway inhibition (IC 50 = 0.03 μM). [10] Tan and coworkers reported the quantitative structure-activity relationship (QSAR) analysis of 54 RAs.…”