Unraveling Regio- and Stereoselectivity in Cyclopropane Fatty Acid Synthase (CFAS) Enzymes across Native and Non-native Lipid Substrates

Abstract Cyclopropane fatty acid synthase (CFAS) enzymes catalyze the formation of cyclopropane rings within bacterial membrane lipids, a transformation implicated in membrane homeostasis, stress adaptation, and antimicrobial resistance. Despite decades of biochemical investigation, the molecular determinants governing CFAS regioselectivity, stereoselectivity, chain specificity, and substrate scope remain poorly understood. Here, we systematically investigate how enzyme orthology, lipid structure, and membrane environment shape CFAS selectivity. Comparative analysis of CFAS orthologues from Escherichia coli, Lactobacillus acidophilus, and Streptococcus thermophilus reveals that the extent of mono- versus di-cyclopropanation, regioselectivity, and stereochemical outcome are not conserved properties but diverge substantially across the enzyme family. A simple two-carbon variation in phospholipid acyl chain length (C16 versus C18) reverses the absolute configuration of the cyclopropane product, revealing an unrecognized structural determinant of stereochemical outcome. Furthermore, we demonstrate that CFAS catalysis extends beyond native phospholipids to synthetic pseudo-phospholipids bearing non-phosphate anionic headgroups and to fatty acids embedded within lipid vesicles, substantially expanding the substrate scope of this enzyme family. Together, these findings provide insight into the molecular basis of CFAS selectivity, advance the understanding of bacterial lipid cyclopropanation, and broaden the synthetic potential of these enzymes for the stereoselective preparation of non-natural cyclopropyl lipids.