title: 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 includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant ofBacillus subtilisQueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, 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 inEscherichia colisuggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.
authors.affiliation: Graduate College of Biomedical Sciences, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766, USA. amohammad@westernu.edu.
authors.forename: Adeba
authors.initials: A
authors.lastname: Mohammad
authors.affiliation: Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207, USA. bonramos@pdx.edu.
authors.forename: Adriana
authors.initials: A
authors.lastname: Bon Ramos
authors.affiliation: Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207, USA. bowklee@gmail.com.
authors.forename: Bobby W K
authors.initials: BW
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207, USA. cohens@oregonstate.edu.
authors.forename: Spencer W
authors.initials: SW
authors.lastname: Cohen
authors.affiliation: Graduate College of Biomedical Sciences, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766, USA. keemia@gmail.com.
authors.forename: Maryam K
authors.initials: MK
authors.lastname: Kiani
authors.affiliation: Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207, USA. iwatard@pdx.edu.
authors.forename: Dirk
authors.initials: D
authors.lastname: Iwata-Reuyl
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University 5500 Campanile Drive, San Diego, CA 92182, USA. bog.stec.2010@gmail.com.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Stec
authors.affiliation: Graduate College of Biomedical Sciences, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766, USA. mswairjo@mail.sdsu.edu.|Department of Chemistry and Biochemistry, San Diego State University 5500 Campanile Drive, San Diego, CA 92182, USA. mswairjo@mail.sdsu.edu.
authors.forename: Manal A
authors.initials: MA
authors.lastname: Swairjo
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009704:Nucleoside Q / Q000096:biosynthesis*; D010084:Oxidation-Reduction; D010802:Phylogeny; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D050505:Mutant Proteins; D009704:Nucleoside Q; D003545:Cysteine
keywords: oxidoreductase; tRNA modification; tunneling fold