Melissa Salmon, Ramesha B. Thimmappa, Robert E. Minto, Rachel E. Melton, Richard K. Hughes, Paul E. O’Maille, Andrew M. Hemmings, and Anne Osbourn

Significance

The triterpenes are a large and highly diverse group of plant natural products. They are synthesized by cyclization of the linear isoprenoid 2,3-oxidosqualene into different triterpene scaffolds by enzymes known as triterpene synthases. This cyclization process is one of the most complex enzymatic reactions known and is only poorly understood. Here, we identify a conserved amino acid residue that is critical for both product and substrate specificity in triterpene synthases from diverse plant species. Our results shed new light on mechanisms of triterpene cyclization in plants and open up the possibility of manipulating both the nature of the precursor and product specificity, findings that can be exploited for the production of diverse and novel triterpenes.

Abstract

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.

See: http://www.pnas.org/content/113/30/E4407.full

PNAS July 26 2016; vol.113; no.30:  E4407–E4414

Fig. 1.

Characterization of sad1 mutants. (A) Biosynthesis of phytosterols and avenacin A-1 in oat. (B–D) RT-PCR analysis of mutant sad1 transcript levels in mRNA extracted from the roots of wild-type (WT) oats and predicted premature termination of translation (B), splicing error (C), and amino acid substitution (D) mutants (Table 1). The oat glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a control. (E) Analysis of protein extracts from root tips of WT and mutant oat lines. (Left) Replicate gel stained with InstantBlue showing protein loading. (Right) Western blot analysis of extracts from predicted amino acid substitution mutants probed with antisera raised against SAD1. A single band of ∼86 kDa corresponding to full-length SAD1 protein is present in the WT and mutants 358, 384, and 1023. (F) Locations of point mutations in sad1 mutants. A schematic of the WT Sad1 gene is shown at the top in red. Exons are represented by boxes and introns by lines. The location of each mutation within the gene is indicated by a vertical line.