An Antifungal Polycyclic Tetramate Macrolactam, Heat-Stable Antifungal Factor (HSAF), Is a Novel Oxidative Stress Modulator in Lysobacter enzymogenes

Polycyclic tetramate macrolactams (PoTeMs) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeMs have been investigated for potential physiological functions in their producers. Here, we used heat-stable antifungal factor (HSAF), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeMs form complexes with iron ions, with an association constant (Ka) of 2.71x106 M21. The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stress and exhibited markedly increased production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and restored normal growth in the mutants under the oxidative stress. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damage rather than as an iron-acquisition siderophore. The characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. The study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells.