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 of Bacillus subtilis QueF, 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 in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.
Highlights
QueF is the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent nitrile reductase that functions in the biosynthetic pathway of the tRNA-modified nucleoside queuosine (Q, [1]), a 7-deazaguanosine nucleoside found at the wobble position of bacterial and eukaryotic tRNAs possessing the GUN anticodon [2]
T-fold homodecamer of two head‐to‐head facing pentamers, each composed of a cyclic arrangement of domains, exists as a homodimer with two active sites located at the interfaces between the two T-fold monomeric tunnelling fold (T‐fold) subunits, forming a tunnel in the center (Figure 3A) [16]
QueF is protected from irreversible oxidation by a conserved intramolecular disulfide between the catalytic Cys55 and a second cysteine (Cys99) located in a helix lining the active site, and that oxidative inactivation of the enzyme is reversible with thioredoxin
Summary
QueF is the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent nitrile reductase that functions in the biosynthetic pathway of the tRNA-modified nucleoside queuosine (Q, [1]), a 7-deazaguanosine nucleoside found at the wobble position of bacterial and eukaryotic tRNAs possessing the GUN anticodon (those encoding for Tyr, His, Asp and Asn) [2]. B. subtilis QueF, a ~160 amino acid protein with a single T-fold domain, is a homodecamer of two head-to-head facing pentamers, each composed of a cyclic arrangement of monomeric T-fold subunits, forming a tunnel in the center (Figure 3A) [16]. T-fold homodecamer of two head‐to‐head facing pentamers, each composed of a cyclic arrangement of domains, exists as a homodimer with two active sites located at the interfaces between the two T-fold monomeric T‐fold subunits, forming a tunnel in the center (Figure 3A) [16]. Both QueF subfamilies harbor a conserved QueF motif embedded in a helix flanking of QueF enzymes residues responsible for NADPH binding. QueF is protected from irreversible oxidation by a conserved intramolecular disulfide between the catalytic Cys and a second cysteine (Cys99) located in a helix lining the active site, and that oxidative inactivation of the enzyme is reversible with thioredoxin. Bioinformatic and phylogenetic analyses of the two QueF subfamilies reveal a conservation pattern of the disulfide that is consistent with a biological role in adaptation to oxidative stress environments
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