Publications

• Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme.

  Swairjo M.A., Reddy R.R., Lee B., Van Lanen S.G., Brown S., de Crecy-Lagard V., Iwata-Reuyl D., Schimmel P.

  QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Ba ... >> More

  QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 61:945-948(2005) [PubMed] [EuropePMC]

• Three-dimensional structure of hyper-modified nucleoside Q located in the wobbling position of tRNA.

  Yokoyama S., Miyazawa T., Iitaka Y., Yamaizumi Z., Kasai H., Nishimura S.

  The hyper-modified nucleoside Q (queuosine) is exclusively located in the wobbling position of anticodons of tRNATyr tRNAHis, tRNAAsn and tRNAAsp that recognise codons NAUC (ref. 1). Queuosine and its hexose-containing derivatives are widely distributed in microorganisms, animals and plants. We co ... >> More

  The hyper-modified nucleoside Q (queuosine) is exclusively located in the wobbling position of anticodons of tRNATyr tRNAHis, tRNAAsn and tRNAAsp that recognise codons NAUC (ref. 1). Queuosine and its hexose-containing derivatives are widely distributed in microorganisms, animals and plants. We confirm here the chemical structure of queuosine as 7-(3, 4-trans-4, 5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine (Fig. 1). The unique structural features of Q are the unusual cyclopentenediol side chain and the 7-deazaguanine ring. Queuosine is one of the most complicated modified nucleosides found thus far. << Less

  Nature 282:107-109(1979) [PubMed] [EuropePMC]

• Biosynthesis of the modified nucleoside Q in transfer RNA.

  Kuchino Y., Kasai H., Nihei K., Nishimura S.

  During biosynthesis of the modified nucleoside Q, 7-(4,5-DIHYDROXYL-1-1-CYCLOPENTEN-3-YL-AMINOMETHYL)-7-DEAZAGUANOSINE, IN TRNA, the carbon atom at position 8 in precursor molecule guanine was expelled together with the nitrogen atom N-7 in a fashion similar to that in the biosynthesis of the nucl ... >> More

  During biosynthesis of the modified nucleoside Q, 7-(4,5-DIHYDROXYL-1-1-CYCLOPENTEN-3-YL-AMINOMETHYL)-7-DEAZAGUANOSINE, IN TRNA, the carbon atom at position 8 in precursor molecule guanine was expelled together with the nitrogen atom N-7 in a fashion similar to that in the biosynthesis of the nucleoside antibiotic toyocamycin. << Less

  Nucleic Acids Res 3:393-398(1976) [PubMed] [EuropePMC]

• Structure determination of a nucleoside Q precursor isolated from E. coli tRNA: 7-(aminomethyl)-7-deazaguanosine.

  Okada N., Noguchi S., Nishimura S., Ohgi T., Goto T., Crain P.F., McCloskey J.A.

  A precursor of modified nucleoside Q isolated from E. coli methyl-deficient tRNA was determined to be 7-(aminomethyl)-7-deazaguanosine. The structure was deduced by means of its chromatographic and electrophoretic mobilities, and UV and mass spectra, in addition to comparison with the synthesized ... >> More

  A precursor of modified nucleoside Q isolated from E. coli methyl-deficient tRNA was determined to be 7-(aminomethyl)-7-deazaguanosine. The structure was deduced by means of its chromatographic and electrophoretic mobilities, and UV and mass spectra, in addition to comparison with the synthesized authentic compound. The same molecule is also found in tRNA of an E. coli mutant selected for deficient synthesis of modified nucleosides. << Less

  Nucleic Acids Res 5:2289-2296(1978) [PubMed] [EuropePMC]

• Expression and characterization of the nitrile reductase queF from E. coli.

  Moeller K., Nguyen G.S., Hollmann F., Hanefeld U.

  The expression and characterization of a nitrile reductase from Escherichia coli K-12 (EcoNR), a newly discovered enzyme class, is described. This enzyme has a potential application for an alternative nitrile reduction pathway. The enzyme activity towards its natural substrate, preQ(0), was demons ... >> More

  The expression and characterization of a nitrile reductase from Escherichia coli K-12 (EcoNR), a newly discovered enzyme class, is described. This enzyme has a potential application for an alternative nitrile reduction pathway. The enzyme activity towards its natural substrate, preQ(0), was demonstrated and optimal working conditions were found to be at 37°C and at pH 7 with Tris buffer. << Less

  Enzyme Microb. Technol. 52:129-133(2013) [PubMed] [EuropePMC]

• Isolation of Q nucleoside precursor present in tRNA of an E. coli mutant and its characterization as 7-(cyano)-7-deazaguanosine.

  Noguchi S., Yamaizumi Z., Ohgi T., Goto T., Nishimura Y., Hirota Y., Nishimura S.

  One of the E. coli mutants selected for deficiency of modified nucleoside Q was found to contain an analogue of Q and normal guanosine in place of Q. The analogue of Q, designated as preQo, was isolated on a large scale from purified tRNATyr containing preQo. The structure of preQo was determined ... >> More

  One of the E. coli mutants selected for deficiency of modified nucleoside Q was found to contain an analogue of Q and normal guanosine in place of Q. The analogue of Q, designated as preQo, was isolated on a large scale from purified tRNATyr containing preQo. The structure of preQo was determined to be 7-(cyano)-7-deazaguanosine by comparison of its ultraviolet absorption spectra, thin-layer chromatographic mobility and mass spectrum with those of synthetic material. << Less

  Nucleic Acids Res 5:4215-4223(1978) [PubMed] [EuropePMC]

• From cyclohydrolase to oxidoreductase: discovery of nitrile reductase activity in a common fold.

  Van Lanen S.G., Reader J.S., Swairjo M.A., de Crecy-Lagard V., Lee B., Iwata-Reuyl D.

  The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mu ... >> More

  The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes. << Less

  Proc. Natl. Acad. Sci. U.S.A. 102:4264-4269(2005) [PubMed] [EuropePMC]

• Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering.

  Wilding B., Winkler M., Petschacher B., Kratzer R., Egger S., Steinkellner G., Lyskowski A., Nidetzky B., Gruber K., Klempier N.

  Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitri ... >> More

  Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase. << Less

  Chemistry 19:7007-7012(2013) [PubMed] [EuropePMC]

• Mechanistic studies of Bacillus subtilis QueF, the nitrile oxidoreductase involved in queuosine biosynthesis.

  Lee B.W., Van Lanen S.G., Iwata-Reuyl D.

  The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions ba ... >> More

  The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a kcat = 0.66 +/-0.04 min-1, KM (preQ0) = 0.237 +/- 0.045 microM, and KM (NADPH) = 19.2 +/-1.1 microM. Based on sequence analysis and homology modeling we predicted previously that Cys55 would be present in the active site and in proximity to the nitrile group of preQ0. Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ0 was present. Furthermore, titrations of the enzyme with preQ0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an alpha,beta-unsaturated thioimide. Site-directed mutagenesis of Cys55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ0. Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis. << Less

  Biochemistry 46:12844-12854(2007) [PubMed] [EuropePMC]