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, primarily by inhibiting pyridoxal 5'-phosphate (PLP)-dependent enzymes. Recent studies suggest that cycloserine may also target cysteine desulfurases involved in iron-sulfur (Fe-S) cluster biogenesis, but the underlying mechanism remains unclear. This study aims to elucidate the mechanistic details of how cycloserine affects Fe-S cluster assembly and to explore its potential against resistant strains.

The effects of DCS and its enantiomer L-Cycloserine (LCS) on Fe-S cluster biosynthesis were investigated using Escherichia coli (E. coli) IscS as a model cysteine desulfurase. Enzyme activity assays were performed to assess inhibition. Liquid chromatography-mass spectrometry (LC-MS) was employed to analyze changes in the PLP cofactor bound to IscS. Antimicrobial efficacy was tested against the uropathogenic E. coli strain CFT073 and experimentally evolved DCS-resistant strains in synthetic human urine medium.

LCS, but not DCS, inhibited the cysteine desulfurase activity of IscS, leading to impaired Fe-S cluster biosynthesis. Specifically, LC-MS analysis revealed that LCS converts the PLP cofactor bound to IscS to pyridoxamine 5'-phosphate (PMP). Furthermore, LCS significantly suppressed the growth of uropathogenic E. coli CFT073 and retained effectiveness against DCS-resistant strains in synthetic human urine medium.

These findings demonstrate that LCS inhibits Fe-S cluster biosynthesis by targeting the cysteine desulfurase IscS through PLP-to-PMP conversion. The results highlight Fe-S cluster biogenesis as a potential antimicrobial target and suggest that LCS may serve as a promising therapeutic alternative for combating DCS-resistant bacterial infections.