L-Cycloserine, not D-Cycloserine, inhibits IscS-mediated iron–sulfur cluster biosynthesis in Escherichia coli

D-Cycloserine (DCS) is a broad-spectrum antibiotic used to treat drug-resistant tuberculosis. It exerts its antimicrobial effects by inhibiting a variety of pyridoxal 5′-phosphate (PLP)-dependent enzymes. Recent studies indicate that cycloserine targets cysteine desulfurases, which are essential for iron–sulfur (Fe–S) cluster biogenesis. However, the mechanistic details of its effect on Fe–S cluster assembly are unclear. In this study, we demonstrate that L-Cycloserine (LCS), but not its enantiomer DCS, inhibits the cysteine desulfurase activity of Escherichia coli ( E. coli ) IscS, resulting in impaired Fe–S cluster biosynthesis. Liquid chromatography-mass spectrometry (LC–MS) analysis showed that LCS converts the IscS-bound PLP cofactor into pyridoxamine 5′-phosphate (PMP). Importantly, LCS significantly inhibited the growth of the uropathogenic E. coli strain CFT073, and remained effective against experimentally-evolved DCS-resistant strains in synthetic human urine medium. Our work highlights Fe–S cluster biosynthesis as a novel antimicrobial target and proposes LCS as a promising alternative therapeutic agent to counteract DCS resistance.