Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

Abstract

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of the nitrile group of 7‐cyano‐7‐deazaguanine (preQ0) to 7‐aminomethyl‐7‐deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF‐catalyzed reaction involves the formation of a covalent thioimide intermediate with a conserved active site cysteine (Cys55) that is prone to oxidation in vivo under different conditions. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide bond formed between the catalytically active Cys55 and a conserved Cys99 located near the active site. When compared to the substrate‐bound structure, this structure is more symmetric and exhibits major rearrangement of the loops responsible for substrate binding. It has been demonstrated that the disulfide does not play a catalytic role because mutation of Cys99 to Ala/Ser does not compromise enzyme activity. Inactivation of the wild‐type enzyme via peroxidation is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible. This is consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin‐like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide‐thiol exchange system may constitute a general protective mechanism of QueF from oxidative stress in vivo. Docking of QueF onto this enzyme complex sheds structural light on the mechanism of redox control and may inform future mutagenesis experiments.Support or Funding InformationThis project was supported by National Science Foundation grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo, National Institutes of Health (NIH) grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, and NIH grant GM70641 to D. Iwata‐Reuyl. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE‐AC02‐76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research, by the NIH, and the National Institute of General Medical Sciences (NIGMS) (including grant P41GM103393).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.