Regulation of and mechanistic insights into terpene biosynthesis

• Applicant: Professor Dr. Michael Groll
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542938137

Terpenes are the largest class of natural products and of immense biological importance. Despite their chemical diversity, they are composed of only two isomeric building blocks dimethylallyl diphosphate (DMAPP, C5) and isopentenyl diphosphate (IPP, C5). In our proposed research, Project 1 deals with isoprenyl diphosphate synthases (IDS) that condense DMAPP with different numbers of IPP molecules to C5n precursors and are classified according to the chain length of their end products. Strikingly, Phaedon cochleariae (Pc) uses IDS1 to regulate the flux of terpene building blocks in response to metal ions to provide defense compounds via geranyl diphosphate (GPP, C10) in the larval stage and farnesyl diphosphate (FPP, C15) for hormone synthesis during metamorphosis. However, the beetle also has additional IDS enzymes that share more than 80% sequence identity with PcIDS1, but differ in the ligand-binding channel as well as the molecular switch that integrates substrate, product and metal ion specificity. Beside PcIDS enzymes, geranylgeranyl (GGPPS, C20) and geranylfarnesyl (GFPPS, C25) diphosphate synthases are of particular interest. Remarkably, GFPPS builds from DMAPP and four sequentially attached IPP molecules a C25 isoprene unit that serves as starter unit for a new class of sesterterpene natural products. For each IDS, we will study the allosteric regulation by divalent metal ions and the intrinsic mechanisms that determine the chain length of the reaction product. Project 2 focuses on the biosynthesis of condensed hydrocarbons that form complex isoprenoid scaffolds. In particular, we aim to get structural and mechanistic insights into the intramolecular cyclization cascade of class I terpene cyclases (TCs), one of the most complex enzymatic reactions in nature. Using chemically synthesized substrates and surrogates, we will elucidate the distinct reaction pathways of sesquiterpenes such as muurolol, cubenol, germacrene A, isoafricanol, pristinol and isoishwarane. X-ray crystallographic studies will be complemented by enzyme activity assays, and product pattern formation will be analyzed by GC/MS and NMR techniques. In addition, theoretical calculations on the energetics of possible reaction pathways will help to develop innovative concepts for enzyme engineering. Based on these results, site-directed mutagenesis experiments are envisioned to design TCs for future semisynthetic applications.

• DFG Programme: Research Grants