Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra, Sanghamitra; Holz, Richard C.

doi:10.1074/jbc.m604117200

Cited by 62 publications

(105 citation statements)

References 44 publications

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“…Considering the similarity between isonitriles and nitriles, nitrile hydration is likely to proceed in a similar manner (Fig. 6B) (33). However, the corresponding residues of ReNHase were unchanged during our investigations (Fig.…”

Section: Time-resolved X-ray Crystallography Of the Reaction Of Renhamentioning

confidence: 53%

Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

Hashimoto

Suzuki

Taniguchi

et al. 2008

Journal of Biological Chemistry

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Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteinesulfinic acid and cysteine-sulfenic acid, catalyzing the hydration of nitriles to amides. Recently, we found that the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking advantage of the slow reactivity of tBuNC and the photoreactivity of nitrosylated NHase, we present the first structural evidence for the catalytic mechanism of NHase with time-resolved x-ray crystallography. By monitoring the reaction with attenuated total reflectanceFourier transform infrared spectroscopy, the product from the isonitrile carbon was identified as a CO molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and stopped by flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron center and then coordinated to the iron ion at 120 min. At 440 min, the electron density of tBuNC was significantly altered, and a new electron density was observed near the isonitrile carbon as well as the sulfenate oxygen of ␣Cys 114. These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by ␣Cys -SOH.Nitrile hydratases (NHases) 2 catalyze the hydration of nitriles to the corresponding amides and are used as catalysts in the production of acrylamide, making them one of the most important industrial enzymes (1, 2). NHases contain a nonheme Fe 3ϩ or non-corrin Co 3ϩ catalytic center. Iron-type NHases show unique photoreactivity; the enzyme is inactivated by nitrosylation in the dark and immediately reactivated by photo-induced denitrosylation (3-5). The protein structure is highly conserved among all known NHases (6 -9) as well as a related enzyme, thiocyanate hydrolase (10). The metal site is also conserved, with a distorted octahedral geometry. All ligand residues are involved in a strictly conserved motif of the ␣ subunit, Cys 1 -Xaa-Leu-Cys 2 -Ser-Cys 3 , where two amide nitrogens of Ser and Cys 3 and three Cys sulfurs are coordinated to the metal (6). Cys 2 and Cys 3 are post-translationally modified to cysteine-sulfinic acid and cysteine-sulfenic acid, respectively (7), which probably take deprotonated forms at the metal site (11). The sixth ligand site is occupied by a solvent molecule (8) or by a NO molecule in nitrosylated iron-type NHase (7).Several reaction mechanisms have been proposed based on the protein structures (1, 6). First, nitriles directly bind to the metal to facilitate the nucleophilic attack of a water molecule on the nitrile carbon. In the other mechanisms, a water molecule activated by the metal directly or indirectly attacks nitriles trapped near the metal. In all cases, the metal is suspected to function as a Lewis acid. By reconstituting iron-type NHase from recombinant unmodified subunits, we demonstrated that the post-translational ...

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Section: Time-resolved X-ray Crystallography Of the Reaction Of Renhamentioning

confidence: 53%

Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

Hashimoto

Suzuki

Taniguchi

et al. 2008

Journal of Biological Chemistry

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“…Product release is rate-limiting and assigned k 3 . Product release was previously shown to be rate-limiting under steady state conditions for both iron-type and cobalt-type NHase enzymes (15,16). Concentration profiles for the progress of the reaction confirm a three-step reaction model (Fig.…”

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confidence: 54%

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Gumataotao

Kuhn

Hajnas

et al. 2013

Journal of Biological Chemistry

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Background:No direct evidence exists for the direct coordination of nitrile to the Fe 3ϩ active site in nitrile hydratases. Results: The first Fe 3ϩ -nitrile intermediate species is reported using stopped-flow spectroscopy. Conclusion: These data establish that the direct ligation of the nitrile substrate occurs during catalytic turnover. Significance: Understanding the catalytic mechanism of nitrile hydratases is critical to harness their bioremediation and industrial potential.

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“…The iron could bind the NNG carboxylate, stabilizing the Michaelis complex and enforcing substrate selectivity. Alternatively, iron could bind the nitramine group, facilitating the elimination of nitrite and the hydrolysis of the imine intermediate as proposed for nitrile hydratase enzymes (45,46). Finally, the iron could coordinate with the nitrite leaving group to facilitate its expulsion, similar to the elimination step in aconitase (47).…”

Section: Discussionmentioning

confidence: 99%

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N -Nitroglycine by Variovorax sp. Strain JS1663

Mahan

Zheng

Fida

et al. 2017

Appl Environ Microbiol

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Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-sporeforming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (nnlA) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed ␤-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the NON bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives. IMPORTANCEThe production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N-Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei. This study reports the isolation of a Gram-negative soil bacterium, Variovorax sp. strain JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a ␤-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help identify other enzymes that cleave the NON bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives.

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Unraveling the Catalytic Mechanism of Nitrile Hydratases

Cited by 62 publications

References 44 publications

Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N -Nitroglycine by Variovorax sp. Strain JS1663

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