Revealing Acyl Chain Selectivity of Cis-to-Trans Isomerase through Profiling of C═C Geometry and Location Isomers of Bacterial Lipids

Bacteria adapt to environmental stress by modifying their membrane lipid structures, including the C═C geometry. Profiling of bacterial lipids with accurate C═C geometry assignment is challenging due to the lack of standards and interference from C═C location isomers. By leveraging two radical reactions: thiyl radical-catalyzed C═C isomerization and the Paternò-Büchi (PB) reaction, we developed an analytical workflow to profile C═C geometric and location isomers in bacterial lipidomes. The high yield (∼80%) of <i>cis</i> (<i>Z</i>)-to-<i>trans</i> (<i>E</i>) C═C isomerization catalyzed by thiyl radicals allows for on-demand synthesis of commercially unavailable lipid C═C geometric isomers. By comparing the retention behavior of <i>Z</i> vs <i>E</i> isomers from reversed-phase liquid chromatography-mass spectrometry (RPLC-MS), we can determine C═C geometry at sub-nM levels. The location of C═C can be further obtained by conducting an online acetone PB reaction after RPLC separation. Applying this workflow to <i>Pseudomonas putida</i>, we profiled 60 lipid species across six subclasses, including the rarely reported glucosaminyl phosphatidylglycerol. We found that both <i>Z</i> and <i>E</i> isomers were present in bacterial lipids, however, with an increase in <i>E</i> isomers after toluene exposure, which correlated with an upregulation of <i>cis</i>-to-<i>trans</i> isomerase (Cti). Our workflow further revealed the chain selectivity of Cti, with a preference for C16:1(<i>n</i>-7Z) > C18:1(<i>n</i>-7Z) > C18:1(<i>n</i>-9Z). This finding provides valuable insights into the dynamics of lipid metabolism during bacterial stress responses.