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

Authors

Irene Anyango Ogutu, Marquette University
Martin St. Maurice, Marquette UniversityFollow
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Publication Date

11-2021

Publisher

MDPI

Source Publication

Catalysts

Source ISSN

2073-4344

Abstract

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 species: one was the apoform (αSer^A) that exhibited no observable activity, and the second (αSer^B) contained its full complement of cobalt ions and was active with a k_cat value of 63 ± 3 s⁻¹ towards acrylonitrile at pH 7.5. The X-ray crystal structure of was determined at 1.85 Å resolution and contained no detectable cobalt per α₂β₂ 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 PtNHase, but were able to hydrate acrylonitrile with k_cat values of 120 ± 6, 29 ± 3, and 14 ± 1 s⁻¹ 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.

Comments

Published version. Catalysts, Vol. 11, No. 11 (November, 2021): 1381. DOI. This article is © authors. Licensee MDPI, Basel, Switzerland. Used with permission. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/4.0/).

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Ogutu, Irene Anyango; St. Maurice, Martin; Bennett, Brian; and Holz, Richard C., "Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095" (2021). Physics Faculty Research and Publications. 181.
https://epublications.marquette.edu/physics_fac/181