Investigations on the Biosynthetic Enzymes for the Earthy Odorant 2-Methylisoborneol

• Applicant: Professor Dr. Jeroen Dickschat
• Subject Area: Organic Molecular Chemistry - Synthesis and Characterisation
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469042295

This project aims at a deep enzyme mechanistic investigation of the 2-MIB biosynthetic enzymes GPPMT and 2-MIBS. These biocatalysts shall also be made available for the enzymatic synthesis of novel 2-MIB analogs by expanding their substrate tolerance systematically.First, several DMAPP and IPP analogs will be obtained by chemical synthesis. Their conversion into GPP analogs by FPPS will be investigated, followed by testing their conversion by GPPMT and then 2-MIBS. Also enzymatically prepared SAM derivatives will be used to further expand the accessible chemical space. In successful cases the products will be purified from preparative scale incubations and their structures will be determined by NMR.A luminescence based method will be developed to efficiently follow terpene synthase activity. Terpene cyclizations lead to diphosphate as a coupled byproduct that can be converted with adenosine 5’-phosphosulfate (APS) by sulfate adenylyltransferase into ATP, followed by coupling to the light generating reaction by firefly luciferase (as used in pyrosequencing). The setup will be optimized to allow for the easy determination of terpene synthase enzyme kinetics, especially of 2-MIBS. For this purpose enzyme concentrations need to be determined for which a fluorescence method using enzyme fusions to the green fluorescent protein will be used.Site-directed mutagenesis of highly conserved residues in GPPMT and 2-MIBS will be performed to study these enzymes mechanistically. For active enzyme variants kinetics will be determined by the luciferase system and compared to wildtype activity. Directed evolution will be used to expand the active site cavities and substrate tolerances of GPPMT and 2-MIBS to make these enzymes accessible for larger substrates. An optimized 2-MIBS variant will be used to produce 2-MIB with a reactive anchor such as a propargyl group that can be used in click chemistry. This makes it possible to connect the obtained 2-MIB analog via a linker to biotin, making the whole molecule immobilizable on streptavidin sepharose, which can then be used to fish out the molecular protein targets from cell lysates. This will be done with selected bacterial 2-MIB producers to investigate if 2-MIB has a molecular target in the producers themselves.

• DFG Programme: Research Grants