A novel thermostable nitrile hydratase

Pereira, Rui; Graham, Daniel L.; Rainey, Fred A.; Cowan, Don A.

doi:10.1007/s007920050078

Cited by 65 publications

(57 citation statements)

References 35 publications

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“…Generally, the mesophilic NHases have exhibited maximum activity near ambient temperature between 20 and 40°C [14,15]. However, thermophilic NHases of Bacillus RAPc8 [16] and Pseudonocardia thermophila [17] exhibited maximum activity at 60°C. The half-life of NHase of R. rhodochrous PA-34 was 2 h at 40°C, 0.5 h at 50°C and it was rapidly inactivated at temperature above 50°C.…”

Section: Effect Of Temperature On Activity and Stability Of Nhasementioning

confidence: 99%

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

2009

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A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purifi ed from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purifi cation of NHase was achieved with 52% yield. The SDS-PAGE of the purifi ed enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 μmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO 3 , Pb(CH 3 COO) 2 and HgCl 2 inhibited the NHase to extent of 89-100%.

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Section: Effect Of Temperature On Activity and Stability Of Nhasementioning

confidence: 99%

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

2009

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“…Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using 18 O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles.…”

mentioning

confidence: 80%

“…1, B and C, with actual NHase enzymes. In 18 O-labeled water, the mechanism shown in Fig. 1B predicts that a single turnover should result in a product with no isotopic enrichment as the [ 16 O]oxygen originally from the sulfenato ligand is used and not the bulk solvent.…”

Section: Nitrile Hydratases (Nhases)mentioning

confidence: 99%

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

Nelp

Song

Wysocki

et al. 2016

Journal of Biological Chemistry

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Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using 18 O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles. Nitrile hydratases (NHases)2 catalyze the formation of amides from nitriles in aqueous, pH-neutral environments without any carboxylic acid byproducts (1, 2). These bacterial enzymes utilize trivalent iron or cobalt metal cations in this non-redox reaction, and uniquely, these metal ions are ligated directly to the protein backbone through amidate ligands and three cysteine sulfur ligands, of which two are post-translationally modified to the sulfinic and sulfenic acid ( Fig. 1A) (3). The mechanism of hydration is slowly emerging through multiple studies and appears to involve the sulfenic acid oxygen as the nucleophile. This sulfenate oxygen is proposed to initially attack the metal-bound nitrile carbon atom, creating a cyclic intermediate that is then resolved through attack at the sulfenic sulfur either directly by water or from the neighboring axial thiolato sulfur (Fig. 1B) (4 -6). Alternative mechanisms have also found support from time-resolved crystallographic and kinetic studies that involve an activated water molecule attacking the metal-bound nitrile (Fig. 1C) (7, 8).Early studies on sulfenato ligands in cobalt complexes demonstrated the nucleophilicity of the sulfenato oxygen (9, 10), and recently, this same characteristic was observed in NHases wherein the sulfenato oxygen reacted with electrophilic boronic acids (11). Quite tellingly, the activities of NHase, the related thiocyanate hydrolase, and NHase mimics were found to depend on the presence of these sulfenato ligands where oxidation to the sulfinate abrogated activity (12-14).To find direct evidence of either a protein-based or a solventbased nucleophile, we became interested in the observations made by Heinrich et al. (14) with an iron NHase mimic bearing two sulfenato ligands that is capable of hydrating nitriles with up to 50 turnovers. They discovered that labeled oxygen from solvent water was exchanged into one of these sulfenato groups only if the nitrile hydration reaction occurred. This provides a...

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“…He reported the purification, characterization, and applications of two hydratases and a nitrilase from two thermophilic Bacillus strains (1,2,19,36,74). Each of the enzymes was more thermostable than corresponding enzymes isolated from mesophiles, but their temperature optima were marginally lower than the growth temperatures of the bacterial strains from which they were isolated (55 to 65°C).…”

Section: Biocatalysismentioning

confidence: 99%