Mutational Study on αGln90 of Fe-Type Nitrile Hydratase from Rhodococcus sp. N771

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 k cat of αQ90E and αQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on K m 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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