Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095
The strictly conserved αSer162 residue in the Co-type nitrile hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 (<i>Pt</i>NHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein spe...
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MDPI AG
2021-11-01
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| author | Irene R. A. M. Ogutu Martin St. Maurice Brian Bennett Richard C. Holz |
| author_facet | Irene R. A. M. Ogutu Martin St. Maurice Brian Bennett Richard C. Holz |
| author_sort | Irene R. A. M. Ogutu |
| collection | DOAJ |
| description | The strictly conserved αSer162 residue in the Co-type nitrile hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 (<i>Pt</i>NHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSer<sup>A</sup>) that exhibited no observable activity, and the second (αSer<sup>B</sup>) contained its full complement of cobalt ions and was active with a <i>k</i><sub>cat</sub> value of 63 ± 3 s<sup>−1</sup> towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSer<sup>A</sup> was determined at 1.85 Å resolution and contained no detectable cobalt per α<sub>2</sub>β<sub>2</sub> heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type <i>Pt</i>NHase, but were able to hydrate acrylonitrile with <i>k</i><sub>cat</sub> values of 120 ± 6, 29 ± 3, and 14 ± 1 s<sup>−1</sup> for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases. |
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| language | English |
| last_indexed | 2024-03-10T05:37:05Z |
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| spelling | doaj.art-22a456b625b1437fa980c0190a0a18e02023-11-22T22:46:24ZengMDPI AGCatalysts2073-43442021-11-011111138110.3390/catal11111381Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095Irene R. A. M. Ogutu0Martin St. Maurice1Brian Bennett2Richard C. Holz3Department of Chemistry, Colorado School of Mines, Golden, CO 80401, USADepartment of Biology, Marquette University, 1428 W. Clybourn St., Milwaukee, WI 53201, USADepartment of Physics, Marquette University, 1420 W. Clybourn St., Milwaukee, WI 53201, USADepartment of Chemistry, Colorado School of Mines, Golden, CO 80401, USAThe strictly conserved αSer162 residue in the Co-type nitrile hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 (<i>Pt</i>NHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSer<sup>A</sup>) that exhibited no observable activity, and the second (αSer<sup>B</sup>) contained its full complement of cobalt ions and was active with a <i>k</i><sub>cat</sub> value of 63 ± 3 s<sup>−1</sup> towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSer<sup>A</sup> was determined at 1.85 Å resolution and contained no detectable cobalt per α<sub>2</sub>β<sub>2</sub> heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type <i>Pt</i>NHase, but were able to hydrate acrylonitrile with <i>k</i><sub>cat</sub> values of 120 ± 6, 29 ± 3, and 14 ± 1 s<sup>−1</sup> for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.https://www.mdpi.com/2073-4344/11/11/1381nitrile hydratasecobalthydrationmutantX-ray crystallographyUV–vis spectroscopy |
| spellingShingle | Irene R. A. M. Ogutu Martin St. Maurice Brian Bennett Richard C. Holz Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 Catalysts nitrile hydratase cobalt hydration mutant X-ray crystallography UV–vis spectroscopy |
| title | Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 |
| title_full | Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 |
| title_fullStr | Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 |
| title_full_unstemmed | Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 |
| title_short | Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 |
| title_sort | examination of the catalytic role of the axial cystine ligand in the co type nitrile hydratase from i pseudonocardia thermophila i jcm 3095 |
| topic | nitrile hydratase cobalt hydration mutant X-ray crystallography UV–vis spectroscopy |
| url | https://www.mdpi.com/2073-4344/11/11/1381 |
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