tailieunhanh - Báo cáo khóa học: Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding

Mutants of a cobalt-containing nitrile hydratase (NHase, EC ) fromPseudonocardia thermophilaJCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected fromthe structure of the substrate binding pocket, the wild-type enzyme showed significantly lower KmandKi values for aromatic substrates and inhibitors, respectively, than alipha-ticones. | Eur. J. Biochem. 271 429-438 2004 FEBS 2003 doi Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding Akimasa Miyanaga1 Shinya Fushinobu1 Kiyoshi Ito2 Hirofumi Shoun1 and Takayoshi Wakagi1 1Department of Biotechnology The University of Tokyo Japan 2Life Science Laboratory Mitsui Chemicals Inc. Togo Mobara-shi Chiba Japan Mutants of a cobalt-containing nitrile hydratase NHase EC 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 Km and K 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 bTyr68 formed hydrogen bonds with both n-butyric acid and aSer112 which is located in the active center. The bY68F mutant showed an elevated Km value and a significantly decreased kcat 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 aCys108 and aCys113 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 aT109S and aY114T were constructed each residue being replaced with an iron-containing one. The aT109S mutant showed similar characteristics to the wild-type enzyme. However the aY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme and oxidative modifications of aCys111 and aCys113 residues were not observed. The aTyr114 residue may be involved in the .