Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095

The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), 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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Autores principales:	Irene R. A. M. Ogutu, Martin St. Maurice, Brian Bennett, Richard C. Holz
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	nitrile hydratase cobalt hydration mutant X-ray crystallography UV–vis spectroscopy Chemical technology TP1-1185 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/22a456b625b1437fa980c0190a0a18e0
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spelling	oai:doaj.org-article:22a456b625b1437fa980c0190a0a18e02021-11-25T17:06:34ZExamination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 309510.3390/catal111113812073-4344https://doaj.org/article/22a456b625b1437fa980c0190a0a18e02021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4344/11/11/1381https://doaj.org/toc/2073-4344The 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.Irene R. A. M. OgutuMartin St. MauriceBrian BennettRichard C. HolzMDPI AGarticlenitrile hydratasecobalthydrationmutantX-ray crystallographyUV–vis spectroscopyChemical technologyTP1-1185ChemistryQD1-999ENCatalysts, Vol 11, Iss 1381, p 1381 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	nitrile hydratase cobalt hydration mutant X-ray crystallography UV–vis spectroscopy Chemical technology TP1-1185 Chemistry QD1-999
spellingShingle	nitrile hydratase cobalt hydration mutant X-ray crystallography UV–vis spectroscopy Chemical technology TP1-1185 Chemistry QD1-999 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
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.
format	article
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
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_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_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_sort	examination of the catalytic role of the axial cystine ligand in the co-type nitrile hydratase from <i>pseudonocardia thermophila</i> jcm 3095
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/22a456b625b1437fa980c0190a0a18e0
work_keys_str_mv	AT ireneramogutu examinationofthecatalyticroleoftheaxialcystineligandinthecotypenitrilehydratasefromipseudonocardiathermophilaijcm3095 AT martinstmaurice examinationofthecatalyticroleoftheaxialcystineligandinthecotypenitrilehydratasefromipseudonocardiathermophilaijcm3095 AT brianbennett examinationofthecatalyticroleoftheaxialcystineligandinthecotypenitrilehydratasefromipseudonocardiathermophilaijcm3095 AT richardcholz examinationofthecatalyticroleoftheaxialcystineligandinthecotypenitrilehydratasefromipseudonocardiathermophilaijcm3095
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Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from <i>Pseudonocardia thermophila</i> JCM 3095

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