D₂O Labeling to measure active biosynthesis of natural products in medicinal plants

Abstract

Plant natural products have served as a prominent source of medicines throughout human history, and are still used today as clinically-approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow-growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically-relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids, and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short time scales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis

“…Thus, we focused our discovery efforts in this species. Though HupA accumulates to a reasonably high level in all tissues within the plant, we had previously established through D 2 O labeling studies that de novo HupA biosynthesis is specific to new growth in the shoots of P. tetrastichus (35). We confirmed through D 2 O labeling studies that active biosynthesis of HupA is only found within the new growth of the shoot (although other primary metabolites are actively made throughout the shoot) (35).…”

“…The specific biosynthetic activity in new growth (35), accompanied by our transcriptomic data, suggests developmental control of Lycopodium alkaloid biosynthesis. Tissue-and cell-specific production of plant alkaloids is well-documented (10,68,69), and the production of bioactive alkaloids within new growth supports a role for these molecules in defending these susceptible new tissues from herbivores.…”

“…Though HupA accumulates to a reasonably high level in all tissues within the plant, we had previously established through D 2 O labeling studies that de novo HupA biosynthesis is specific to new growth in the shoots of P. tetrastichus (35). We confirmed through D 2 O labeling studies that active biosynthesis of HupA is only found within the new growth of the shoot (although other primary metabolites are actively made throughout the shoot) (35). These experiments also revealed that HupA labeling was more extensive in the leaves of this region compared to the stem, suggesting an enrichment of biosynthetic gene expression here (Fig.…”

“…D 2 O Labeling to Identify Active Metabolism. D 2 O labeling studies were performed much as previously described (35). Additional details are available in SI Appendix.…”

The chloroalkaloid (−)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants

“…The resulting transcripts were annotated by BLAST search against the UniProtKB/Swiss-Prot database, and their tissue-specific abundance was quantified by transcripts per million (TPM) values. Using D 2 O-labeling technique, a previous study explored the plausible (−)-acutumine biosynthetic pathway by tracing deuterium-labeled benzylisoquinoline alkaloids (BIAs) in M. canadense 25 . Based on this proposed pathway and prior knowledge about BIA metabolism in other plants, we examined functionally annotated transcripts that are highly and differentially expressed in the root versus the leaf or stem tissue of M. canadense, from which numerous candidate genes for the biosynthesis of (−)-acutumine and other related BIAs were identified ( Fig.…”