Insights into E. coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Abstract Cyclopropane fatty acid synthases (CFAS) are a class of S ‐adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene‐based approaches. This work describes the characterisation of E. coli CFAS ( ec CFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ec CFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ec CFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ec CFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ec CFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ec CFAS with the SAM regenerating At HMT enzyme in the presence of CH 3 I and CD 3 I respectively.