Abstract
The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ(0)) to 7-aminomethyl-7-deazaguanine (preQ(1)), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ(0), trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ(0) bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces. In both structures, a preQ(0) molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. Both structural and transient kinetic data show that preQ(0) binding, not thioimide formation, induces a large conformational change in and closure of the active site. Based on these data, we propose a mechanism for the activation of the Cys-55 nucleophile and subsequent hydride transfer.
Highlights
QueF is the only nitrile reductase known in biology
In 2005, we reported the discovery of a new dehydrogenase enzyme family involved in nitrile metabolism, QueF (4, 5), which catalyzes the NADPH-dependent reduction of a nitrile group to a primary amine in the biosynthesis of the tRNAmodified nucleoside queuosine (Q),4 a structurally complex nucleoside found at the wobble position of tRNAs possessing the GUN anticodon
QueF enzymes had been misannotated in genomic databases as GTP cyclohydrolase I (GCYH-I)like enzymes (5)
Summary
QueF is the only nitrile reductase known in biology. Results: x-ray crystallography and transient kinetics provide evidence for a thioimide adduct and its rate of formation. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces In both structures, a preQ0 molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. QueF catalyzes the final step of the tRNA-independent portion of the Q pathway, the 2-fold reduction of the advanced intermediate 7-cyano-7-deazaguanine (preQ0) to preQ1 This reaction constitutes the only example of biologically relevant nitrile reduction identified to date and represents a new biochemical transformation. The importance of nitrile reduction in industrial chemical processes and the unique activity of QueF have positioned it as a potential new candidate for industrial biocatalysis Based on their amino acid sequences, QueF enzymes are proposed to belong to the tunneling-fold (T-fold) superfamily (8), a structural superfamily of functionally distant proteins that bind planar pterin/purine substrates.
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