Chemoproteomics Reveals Disruption of Metal Homeostasis and Metalloproteins by the Antibiotic Holomycin

The natural product holomycin contains a unique cyclic
ene–disulfide
and exhibits broad-spectrum antimicrobial activities. Reduced holomycin
chelates metal ions with a high affinity and disrupts metal homeostasis
in the cell. To identify cellular metalloproteins inhibited by holomycin,
reactive-cysteine profiling was performed using isotopic tandem orthogonal
proteolysis–activity-based protein profiling (isoTOP-ABPP).
This chemoproteomic analysis demonstrated that holomycin treatment
increases the reactivity of metal-coordinating cysteine residues in
several zinc-dependent and iron–sulfur cluster-dependent enzymes,
including carbonic anhydrase II and fumarase A. We validated that
holomycin inhibits fumarase A activity in bacterial cells and diminishes
the presence of iron–sulfur clusters in fumarase A. Whole-proteome
abundance analysis revealed that holomycin treatment induces zinc
and iron starvation and cellular stress. This study suggests that
holomycin inhibits bacterial growth by impairing the functions of
multiple metalloenzymes and sets the stage for investigating the impact
of metal-binding molecules on metalloproteomes by using chemoproteomics.