Covalent cysteine modification by reactive oxygen species (ROS) has been implicated in regulating diverse biological processes, yet global understanding of this modification has remained fragmentary. Developing new approaches for detecting cysteine modification, Xiao et al. ( 2020) recently charted a comprehensive map of cysteine oxidation across tissues and life stages.
Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na ϩ -pumping NADH:quinone oxidoreductase (Na ϩ -NQR.) We show that although korormicin is an inhibitor of Na ϩ -NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O 2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na ϩ -NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na ϩ -NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na ϩ -NQRs confers resistance against korormicin. This is likely the reason that a functional Na ϩ -NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora. IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na ϩ -pumping NADH:quinone oxidoreductase (Na ϩ -NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na ϩ -NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na ϩ -NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O 2 . FIG 2 Na ϩ -NQR electron transfer scheme illustrating probable sites of side reactions with O 2 in the presence of korormicin (green). The structure model was drawn in Swiss-PdbViewer; the structure was obtained from PDB 4P6V. FIG 3 Structural origin of korormicin sensitivity. Sequence alignment of a portion of the NqrB subunit shows the G140 and G141 residues (V. cholerae numbering) present in every known korormicin-sensitive Na ϩ -NQR, with the table below highlighting this relationship.a C...
Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.
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