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Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy. J Biol Inorg Chem 2019 Oct;24(7):1105-1113

Date

09/25/2019

Pubmed ID

31549242

DOI

10.1007/s00775-019-01720-y

Scopus ID

2-s2.0-85073765004 (requires institutional sign-in at Scopus site)   5 Citations

Abstract

Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for "green" chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation.

Author List

Lankathilaka KPW, Stein N, Holz RC, Bennett B

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Anaerobiosis
Electron Spin Resonance Spectroscopy
Hydro-Lyases
Iron
Rhodococcus equi