Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy

Lankathilaka, K.P. Wasantha; Stein, Natalia; Holz, Richard C.; Bennett, Brian

doi:10.1007/s00775-019-01720-y

Cited by 4 publications

(7 citation statements)

References 24 publications

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“…The newly predominant signal, exhibiting ∼50% of the spin density, exhibits a g 1 -resonance at 3050 G. Its features are quite similar to those observed in earlier work upon the addition of acetonitrile to Re NHase, as well as a signal detected upon the addition of acetonitrile to intact Re NHase-expressed in E. coli . In the present work, the signal with g 1 = 3050 is labeled NHase Prod1 to indicate that it arises from a product-bound complex.…”

Section: Resultssupporting

confidence: 83%

“…23 In the present work, the signal with g 1 = 3050 is labeled NHase Prod1 to indicate that it arises from a product-bound complex. The most anisotropic signal in spectra B and C, which was also observed in the earlier work, 23 accounts for <10% of the total spins and exhibits a g 1 -value of 2.276 (i.e., the peak at 2975 G). This signal is very similar to the one displayed by the reversibly inhibited noncovalent complex of NHase with butyric acid, NHase BA (g 1 = 2.281).…”

Section: ■ Resultsmentioning

confidence: 94%

“…NHase was isolated using an anaerobic protocol as previously described in detail . Briefly, the α and β genes were inserted into a pET 28a + plasmid and the ε gene was inserted into a pET 21a + plasmid, and the genes were co-expressed in Escherichia coli BL21 DE3 cells.…”

Section: Methodsmentioning

confidence: 99%

“…NHase was isolated using an anaerobic protocol as previously described in detail. 23 Briefly, the α and β genes were inserted into a pET 28a + plasmid and the ε gene was inserted into a pET 21a + plasmid, and the genes were co-expressed in Escherichia coli BL21 DE3 cells. The active enzyme was isolated using two anion exchange chromatography steps, eluted with NaCl, with an intervening metal affinity chromatography step, eluted with imidazole.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

et al. 2021

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A new method to trap catalytic intermediate species was employed with Fe-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase). ReNHase was incubated with substrates in a 23% (w/w) NaCl/H 2 O eutectic system that remained liquid at −20 °C, thereby permitting the observation of transient species that were present at electron paramagnetic resonance (EPR)detectable levels in samples frozen while in the steady state. Fe III -EPR signals from the resting enzyme were unaffected by the presence of 23% NaCl, and the catalytic activity was ∼55% that in the absence of NaCl at the optimum pH of 7.5. The reaction of ReNHase in the eutectic system at −20 °C with the substrates acetonitrile or benzonitrile induced significant changes in the EPR spectra. A previously unobserved signal with highly rhombic gvalues (g 1 = 2.31) was observed during the steady state but did not persist beyond the exhaustion of the substrate, indicating that it arises from a catalytically competent intermediate. Distinct signals due to product complexes provide a detailed mechanism for product release, the rate-limiting step of the reaction. Assignment of the observed EPR signals was facilitated by density functional theory calculations, which provided candidate structures and g-values for various proposed ReNHase intermediates. Collectively, these results provide new insights into the catalytic mechanism of NHase and offer a new approach for isolating and characterizing EPR-active intermediates in metalloenzymes.

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Section: Resultssupporting

confidence: 83%

Section: ■ Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

et al. 2021

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“…When nitrile materials enter the natural environment, they pose serious threats to the survival of animals and plants, human life and ecological environment. Some wild strains containing NHase have been widely used in the industrial production of acrylamide [5,6], nicotinamide and other amides, which have high industrial value [7][8][9][10]. In the traditional chemical industry, the synthetic production of amides is always performed with harsh reaction conditions and produces a large amount of by-products and pollutants.…”

Section: Introductionmentioning

confidence: 99%

Efficient biodegradation of nitriles by a novel nitrile hydratase derived from Rhodococcus erythropolis CCM2595

Huang

Cui

et al. 2021

Biotechnology & Biotechnological Equipment

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In our previous study, we firstly cloned the gene of a novel ReNHase (NHase from Rhodococcus erythropolis CCM2595), and the strain was shown to degrade only phenol, hydroxybenzoate, p-chlorophenol, aniline and other aromatic compounds. Here in, we further purified the ReNHase from R. erythropolis CCM2595 and detected its properties of biodegradation. We constructed a plasmid with the gene of ReNHase with His-tag. The encoding ReNHase was cloned and overexpressed in recombinant Escherihia coli and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The substrate scope of the new heterologous ReNHase was tested and the efficiency of degradation of nitriles by the ReNHase was also investigated by high efficiency liquid chromatography. We also studied the effect of temperature and pH on the catalysis of adiponitrile by the purified ReNHase. The recombinant E.coli showed high catalytic regioselectivity with high substrate affinity towards dinitriles (especially for adiponitrile) whereas lower affinity towards mononitriles. Compared to whole-cell catalysis, the catalytic time was shortened significantly with the purified ReNHase. The enzyme activity of crude recombinant E.coli was 635 U g −1 (DCW), while the specific activity of purified ReNHase was 63.107 U mg −1 . The apparent Km value for the purified ReNHase is 6.6252 mmol L −1 , which revealed the good affinity between the purified ReNHase and adiponitrile. The reaction Kcat is 82.77 s −1 and Kcat/Km is 1.249 × 10 4 (mol −1 L s −1 ), which showed high catalytic activity towards adiponitrile. We propose that this purified ReNHase may be applied for the industry and sewage treatment for environmental protection.

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Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases

Miller,

Huntoon,

Kaley

et al. 2024

Journal of Inorganic Biochemistry

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Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy

Cited by 4 publications

References 24 publications

Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

Efficient biodegradation of nitriles by a novel nitrile hydratase derived from Rhodococcus erythropolis CCM2595

Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases

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