Melissa Salmon1, Caroline Laurendon1, Maria Vardakou1, Jitender Cheema2, Marianne Defernez3, Sol Green4,Juan A. Faraldos5& Paul E. O’Maille1,6
Salmon, M.et al.Emergence of terpene cyclization in Artemisia annua. Nat. Commun.6:6143 doi: 10.1038/ncomms 7143 (2015)
1 John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK.
2 John Innes Centre, Computational and Systems Biology, Norwich Research Park, Norwich NR4 7UH, UK.
3 Institute of Food Research, Analytical Sciences Unit, Norwich Research Park, Norwich NR4 7UA, UK.
4 Plant and Food Research, 120 Mt Albert Road, Sandringham, Auckland 1025, New Zealand.
5 School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK.
6 Institute of Food Research, Food & Health Programme, Norwich Research Park, Norwich NR4 7UA, UK. Correspondence
and requests for materials should be addressed to P.E.O. (email: firstname.lastname@example.org)
From Paul O'Maille's group comes this advance in understanding terpene cyclization mechanisms. Terpenoids are the largest group of natural products found in nature and many of them derive their biological activities from the presence of one of more rings in their structures. The introduction of rings during the conversion of isoprenyl diphosphate precursors of various sizes into olefinic products is a major source of chemical diversity. The evolution of terpene synthases capable of introducing variable ring structures into their products opened many avenues of natural product biosynthesis for emerging plant species, and so the switch from simple linear chain products into those which underwent cyclization (and subsequently, other structural rearrangements) marked an important development in the evolution of secondary metabolic pathways.
Salmon et al. compared two well known sesquiterpene synthases to identify residues important for cyclizing their linear farnseyl diphosphate (FPP) substrate; (E)-beta-farnesene synthase (EBS), which makes a linear C-15 olefin, and amorpha-4,11-diene synthase, which converts the same substrate into a bicyclic product. They used structure based combinatorial protein engineering (SCOPE) to breed natural mutations into a subset of residues of EBS and screened the mutants by enzyme activity until mutants appeared which produced cyclic products. They found a single amino acid mutation was sufficient to trigger cyclization of FPP and a small number of additional residues play secondary roles in controlling the reaction pathway. This underscores the pivotal role of individual amino acids in controlling the reaction sequence in terpene synthase reactions. A single residue can determine stereochemistry as well as the formation of rings or migration of hydrides or methyl groups, opening the way for a wide multitude of distinct metabolic end products from a small group of evolving catalysts acting on common metabolic intermediates.
The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-beta-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of approx. 27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.