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. 2023 Sep 15;18(9):1909-1914.
doi: 10.1021/acschembio.3c00360. Epub 2023 Aug 10.

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

Andrew N Chan  1 , Xiaoyan Chen  1 , Julia A Falco  2 , Daniel W Bak  2 , Eranthie Weerapana  2 , Bo Li  1   3
Affiliations

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

Andrew N Chan et al. ACS Chem Biol. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
(A) Structures of holomycin and reduced holomycin. (B) Proteomic workflows for protein abundance measurements. (C) Proteomic workflows for quantitative cysteine-reactivity profiling of holomycin-treated proteomes. CuAAC, copper-catalyzed azide–alkyne cycloaddition; LC/LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry. (D) Abundance-corrected isoTOP-ABPP ratio.
Figure 2.
Figure 2.
ReDiMe ratios (R) for each protein identified from two biological replicates: set1 (x axis) and set2 (y axis). Values of log2(R) are shown. Among the top and bottom 20 hits, proteins related to metal homeostasis are highlighted in yellow, and proteins related to redox homeostasis and general stress response are highlighted in pink.
Figure 3.
Figure 3.
(A) Abundance-corrected isoTOP-ABPP ratios of IA–alkyne-labeled peptides from proteomes treated with holomycin (light) and DMSO (heavy). Iron-binding proteins are colored in pink, and zinc-binding proteins are colored in yellow. Map likely binds iron(II) but can also bind zinc. (B) Activity of recombinant FumA purified from cultures treated with DMSO or holomycin. Formation of fumarate from malate catalyzed by FumA was measured based on absorbance at 250 nm. The inset shows FumA purified from a DMSO-treated culture (top) and from a holomycin-treated culture (bottom). This experiment was repeated twice more with comparable results (Figure S3).
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