Probing Sesquitperpene Synthase Chemistry with Non-Canonical Amino Acids
Terpene synthases catalyse complex, multistep reactions that generate thousands of structurally diverse hydrocarbons of biological and commercial importance. Unlike many other biochemical reactions, terpene biosynthesis is essentially carbocationic in nature. Previous work in our lab has revealed many of the mechanistic details of the conversion of FPP by aristolochene synthase (AS), but the key step, the generation of the positively charged eudesmane cation from the uncharged intermediate germacrene A is only poorly understood.
Hence the key unresolved question is how this enzyme achieves the protonation of an isolated double bond to generate what appears to be a high-energy carbocationic intermediate. We propose here to use an approach based on the introduction of natural and non-natural amino acids into the active site of AS to test the proposal that AS uses both an appropriately placed general acid and stabilization of the cationic transition state (and product) through cation-pi interaction to protonate the double bond in a conformationally activated ring system.
Synthesis and introduction into AS of amino acids that carry non-canonical aromatic sides chains will be used to test the function of cation stabilisation, while fluorination of potential general acids will modulate the protonation step during AS catalysis. Since most sesquiterpene cyclases follow similar mechanisms the results obtained here will shed light on intricate details of most of these enzymes that all rely on a shared three-dimensional fold for activity. Hence this work will help decipher the mechanistic principles used by terpene cyclases and may eventually allow us to engineer the specificity of these enzymes in a rational way to get closer to the ultimate goal, namely the expansion of the terpenome through the design of novel (unnatural) terpenoids with many potential applications as pharmaceuticals or agrochemicals.
Diversity Oriented Synthesis of Farnesyl Pyrophosphate Analogues as Mechanistic Probes and as Precursors to Modified Natural Products
Sesquiterpenes are an important class of natural products that exhibit a wide variety of structural variation and biological function with applications ranging from uses as scents and oils to agrochemicals and pharmaceuticals. In contrast to this diversity they are, however, metabolites originating from a single parent compound, farnesyl pyrophospate (FPP). This incredible array of products is generated from FPP by a single class of enzyme – sesquiterpene cyclases – all of which share a common 3D structure.
How this stunning diversity of products is formed from one compound with such remarkable fidelity is a major challenge for modern chemical biology. Each sesquiterpene cyclase enzyme must guide neutral or carbocationic intermediates through a series of specific steps whilst preventing reaction with solvent and rearrangement to undesired by-products of very similar energy and conformation.
In order to examine and solve this problem we intend to synthesise a series of FPP analogue compounds and challenge sesquiterpene cyclase enzymes with them. The synthesis of these compounds is designed in such a way that a combinatorial approach may be used in the future in order to prepare diverse libraries of these materials. In this study the compounds are designed to test the proposed mechanisms by which sesquiterpene cyclases guide FPP and intermediate materials to the sesquiterpene products.
The proposed compounds bear functional groups that are designed to either stabilise or destabilise carbocationic intermediates that have been predicted to form during the formation of sesquiterpene hydrocarbons.
They should therefore give predictable products when tested with our range of sesquiterpene cyclase enzymes if the proposed mechanisms of action are true. If this does not prove to be the case then they will give us information allowing us to propose mechanisms that fit all the observed experimental data. FPP analogues that prove to be substrates for these enzymes will inevitably produce sesquiterpenoid materials of novel structure and function. This will expand the range of biologically active compounds that are available to us and moreover, these compounds, which often have complex structures will have been prepared without tedious and expensive total synthesis.
In summary this project will make great strides in our understanding of terpene cyclase chemistry and also provide a method for the generation of unnatural terpenes with potential commercial applications in both the pharmaceutical and agrochemical industries.
We have recently worked on adapting natural light-sensing proteins to produce new entities. When light of the correct wavelength excites a bound cofactor, a covalent adduct is formed that causes the protein to change conformation. In this new conformation, a previously tightly-bound helix is freed and is able to interact with a partner protein.
De novo designed proteins are not only a rigorous test of our understanding of natural biomacromolecules but they should also shed new light on the molecular mechanisms employed by proteins and allow us to design proteins with functions unprecedented in nature.
• Juan A. Faraldos, Alicja K, Antonczak, Verónica González, Rebecca Fullerton, Eric M. Tippmann and Rudolf K. Allemann J. Am. Chem. Soc., 133 (35), 13906-13609 (2011). DOI:10.1021/ja205927u
• Robert J. Mart, Dilruba Meah and Rudolf K. Allemann ChemBioChem, 17, 698–701 (2016). DOI:10.1002/cbic.201500469
Christopher J. Weston, C. H. Cureton, Melanie J. Calvert, Oliver S. Smart and Rudolf K. Allemann
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