Takarada Hiromi, Kawano Yoshiaki, Hashimoto Kouichi, Nakayama Hiroshi, Ueda Shunsaku, Yohda Masafumi, Kamiya Nobuo, Dohmae Naoshi, Maeda Mizuo, Odaka Masafumi
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
Biosci Biotechnol Biochem. 2006 Apr;70(4):881-9. doi: 10.1271/bbb.70.881.
Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, 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 alphaQ90E and alphaQ90N 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 alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. 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 alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.
来自红球菌属N771的腈水合酶（NHase）是一种非血红素铁酶，具有翻译后修饰的半胱氨酸配体，即αCys112 - SO2H和αCys114 - SOH。我们用谷氨酸或天冬酰胺取代了αGln90，αGln90在所有已知的NHases中都保守，且参与催化中心周围的氢键网络。αQ90E和αQ90N突变体的kcat分别降至野生型的24%和5%，但突变对Km的影响不是很显著。在这两个突变体中，αCys114 - SOH修饰似乎如天然NHase一样负责催化作用。我们使亚硝基化的αQ90N突变体结晶，并以1.43 Å的分辨率确定了其结构。除了突变位点及其附近区域外，该结构与天然亚硝基化NHase的结构基本相同。天然和αQ90N突变体NHases之间的结构差异表明αGln90与铁中心之间的氢键网络对催化活性很重要。
