Mutational Study on .ALPHA.Gln90 of Fe-Type Nitrile Hydratase from Rhodococcus sp. N771

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  • TAKARADA Hiromi
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology Maeda Bioengineering Laboratory, RIKEN
  • KAWANO Yoshiaki
    Protein Crystallography Methodology Team, Advanced Protein Crystallography Research Group, RIKEN Harima Institute
  • HASHIMOTO Kouichi
    Protein Crystallography Methodology Team, Advanced Protein Crystallography Research Group, RIKEN Harima Institute
  • NAKAYAMA Hiroshi
    Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN
  • UEDA Shunsaku
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology
  • YOHDA Masafumi
    Faculty of Technology, Tokyo University of Agriculture and Technology
  • KAMIYA Nobuo
    Protein Crystallography Methodology Team, Advanced Protein Crystallography Research Group, RIKEN Harima Institute
  • DOHMAE Naoshi
    Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN
  • MAEDA Mizuo
    Maeda Bioengineering Laboratory, RIKEN
  • ODAKA Masafumi
    Maeda Bioengineering Laboratory, RIKEN Faculty of Technology, Tokyo University of Agriculture and Technology

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Other Title
  • Mutational Study on αGln90 of Fe-Type Nitrile Hydratase from Rhodococcus sp. N771
  • Mutational Study on アルファ Gln90 of Fe Type Nitrile Hydratase from Rhodococcus sp N771
  • Mutational Study on αGln90 of Fe-Type Nitrile Hydratase from<i>Rhodococcus</i>sp. N771

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Abstract

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, αCys112-SO2H and αCys114-SOH. We replaced αGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of αQ90E and αQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the αCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated αQ90N mutant and determined its structure at a resolution of 1.43 Å. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and αQ90N mutant NHases suggested the importance of the hydrogen bond networks between αGln90 and the iron center for the catalytic activity.

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