Miyanaga Akimasa, Fushinobu Shinya, Ito Kiyoshi, Shoun Hirofumi, Wakagi Takayoshi
Department of Biotechnology, The University of Tokyo, Japan.
Eur J Biochem. 2004 Jan;271(2):429-38. doi: 10.1046/j.1432-1033.2003.03943.x.
Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.
构建了嗜热假诺卡氏菌JCM 3095中参与底物结合、催化和活性中心形成的含钴腈水合酶（NHase，EC 4.2.1.84）的突变体，并研究了它们的特性和晶体结构。正如从底物结合口袋的结构所预期的那样，野生型酶对芳香族底物和抑制剂的K(m)和K(i)值分别明显低于脂肪族底物和抑制剂。在与抑制剂（正丁酸）形成的复合物的晶体结构中，βTyr68的羟基与位于活性中心的正丁酸和αSer112都形成了氢键。βY68F突变体的K(m)值升高，k(cat)值显著降低。不含可检测到的钴原子的脱辅酶是从在无钴离子培养基中生长的大肠杆菌细胞中制备的。它没有可检测到的活性。在脱辅酶结构中形成了αCys108和αCys113之间的二硫键。在半胱氨酸簇区域高度保守的序列基序中，有两个位置在含钴或含铁腈水合酶中是专门保守的。构建了两个突变体（αT109S和αY114T），每个残基都被含铁的残基取代。αT109S突变体表现出与野生型酶相似的特性。然而，与野生型酶相比，αY114T突变体的钴含量和催化活性非常低，并且未观察到αCys111和αCys113残基的氧化修饰。αTyr114残基可能参与了与嗜热假诺卡氏菌腈水合酶激活蛋白的相互作用。
