Bioorthogonal\nMonomycolate of Trehalose Disclosed\nthe <i>O</i>‑Mycoloylation of Mycoloyltransferases\nand Other Cell Envelope Proteins in C. glutamicum

Protein mycoloylation is a recently identified unusual\npost-translational\nmodification (PTM) exclusively observed in Mycobacteriales, an order\nof bacteria that includes several human pathogens. These bacteria\npossess a distinctive outer membrane, known as the mycomembrane, composed\nof very long-chain fatty acids called mycolic acids. It has been demonstrated\nthat a few mycomembrane proteins undergo covalent modification with\nmycolic acids in the model organism Corynebacterium\nglutamicum through the action of mycoloyltransferase\nMytC. This PTM represents the first example of protein <i>O</i>-acylation in prokaryotes and also the first example of protein modification\nby mycolic acid. Many questions about the specificity of protein <i>O</i>-mycoloylation remain crucial for understanding its evolutionary\nsignificance in Mycobacteriales and its role in cell physiology.\nWe have developed the first bioorthogonal mycolate donor featuring\nthe natural mycolic acid pattern, enabling direct, unambiguous transfer\nof the lipid moiety to its acceptors and efficient metabolic labeling\nand enrichment of MytC protein substrates. Mass spectrometry analysis\nof the labeled proteins and comparative proteomic analysis of the\ncell envelope proteome between wild-type and Δ<i>mytC</i> strains identified an unbiased list of 21 proteins likely mycoloylated\nin the cell. The robustness of our approach is demonstrated by the\nsuccessful biological validation of mycoloylation in 6 candidate proteins\nwithin wild-type cells, revealing the characteristic profile of proteins\nmodified with natural mycolates. These findings provide interesting\ninsights into the significance of this new lipidation pathway and\npave the way for understanding their function, especially concerning\nthe mycoloyltransferase family that includes the essential Antigen85\nenzymes in Mycobacteria.