Mutational Study on αGln90 of Fe-Type Nitrile Hydratase from<i>Rhodococcus</i>sp. N771

Takarada, Hiromi; Kawano, Yoshiaki; Hashimoto, Kouichi; Nakayama, Hiroshi; Ueda, Shunsaku; Yohda, Masafumi; Kamiya, Nobuo; Dohmae, Naoshi; Maeda, Mizuo; Odaka, Masafumi

doi:10.1271/bbb.70.881

Cited by 30 publications

(31 citation statements)

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“…In the crystal structure of inactive NHase, several functionally important hydrogen bonds were observed in the catalytic center (14,15,76,78). We were able to identify the same interactions in our models and to check their role in the enzyme dynamics.…”

Section: Functional Hydrogen Bondsmentioning

confidence: 76%

Molecular Dynamics Simulations of the Photoactive Protein Nitrile Hydratase

Kubiak

Nowak

2008

Biophysical Journal

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Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, alpha and beta. Hydrogen bonds between betaArg-56 and alphaCys-114 sulfenic acid (alphaCEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength lambda < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.

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Section: Functional Hydrogen Bondsmentioning

confidence: 76%

Molecular Dynamics Simulations of the Photoactive Protein Nitrile Hydratase

Kubiak

Nowak

2008

Biophysical Journal

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“…The protein in these assays underwent lyophilization to remove natural abundance water and was also incubated for at least an hour after being resuspended in the labeled [ 18 O]water before being added to substrate. Studies using 17 O-labeled water and electron paramagnetic spectroscopy have shown that water molecules in the active site, specifically the active site metal-bound aquo or hydroxo ligand, are easily exchangeable (24,25). Additionally, the active sites of NHases exist at the interface of the ␣-and ␤-subunits (26,27), and because ToyJ in this experiment is monomeric (16), the active site is unlikely to harbor tightly nonexchangeable water molecules as it is probably exposed to the solvent.…”

Section: Figure 2 Analysis Of Toyj Mass Spectrometry Data For Incorpmentioning

confidence: 83%

“…1C predicts that solvent-derived oxygen would be incorporated on the first and all subsequent turnovers and that the protein would never become enriched with solvent-derived oxygen. Single turnover experiments to distinguish between these possibilities are challenging because of the relatively high catalytic efficiency of this class of enzyme with turnover rates for aromatic nitrile substrates ranging from 90 to 159 s Ϫ1 and turnover rates for aliphatic nitrile substrates approaching 1,000 s Ϫ1 and even 4,500 s Ϫ1 for a thermophilic NHase (15)(16)(17)(18).…”

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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“…However, αGln90 is conserved in all known NHases and involved in the hydrogen‐bonding network around the catalytic center. The mutation of this residue resulted in a decrease of the enzymatic activity . Furthermore, βArg52 plays an important role in α subunit exchange and may have a positive effect on the catalytic efficiency .…”

Section: Resultsmentioning

confidence: 99%

Modulation of Nitrile Hydratase Regioselectivity towards Dinitriles by Tailoring the Substrate Binding Pocket Residues

et al. 2017

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The regioselective hydration of dinitriles is one of the most attractive approaches to prepare ω‐cyanocarboxamides or diamides and such regioselectivity is often beyond the capability of chemical catalysts. The use of nitrile hydratase to biotransform dinitriles selectively would be highly desirable. Molecular docking of two aliphatic dinitriles and two aromatic dinitriles into the active site of a nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 allowed the identification of proximal NHase substrate binding pocket residues. Four residues (βLeu48, βPhe51, βTyr68, and βTrp72) were selected for single‐ and double‐point mutations to modulate the NHase regioselectivity towards dinitriles. Several NHase mutants with an altered regioselectivity were obtained, and the best one was Y68T/W72Y. Docking experiments further indicated that the poor binding affinity of aliphatic and aromatic ω‐cyanocarboxamides to the NHase variants resulted in distinct regioselectivity between wild‐type and mutated NHases.

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Mutational Study on αGln90 of Fe-Type Nitrile Hydratase fromRhodococcussp. N771

Cited by 30 publications

References 1 publication

Molecular Dynamics Simulations of the Photoactive Protein Nitrile Hydratase

Molecular Dynamics Simulations of the Photoactive Protein Nitrile Hydratase

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

Modulation of Nitrile Hydratase Regioselectivity towards Dinitriles by Tailoring the Substrate Binding Pocket Residues

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