Nitrile Hydratase Research Articles

Catalytic and post-translational maturation roles of a conserved active site serine residue in nitrile hydratases

A highly conserved second-sphere active site αSer residue in nitrile hydratase (NHase), that forms a hydrogen bond with the axial metal-bound water molecule, was mutated to Ala, Asp, and Thr, in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Thr in the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase). All five mutants were successfully purified; metal analysis via ICP-AES indicated that all three Co-type PtNHase mutants were in their apo-form while the Fe-type αSer117Ala and αSer117Thr mutants contained 85 and 50 % of their active site Fe(III) ions, respectively. The kcat values obtained for the PtNHase mutant enzymes were between 0.03 ± 0.01 and 0.2 ± 0.02 s−1 amounting to <0.8 % of the kcat value observed for WT PtNHase. The Fe-type ReNHase mutants retained some detectable activity with kcat values of 93 ± 3 and 40 ± 2 s−1 for the αSer117Ala and αSer117Thr mutants, respectively, which is ~5 % of WT ReNHase activity towards acrylonitrile. UV–Vis spectra coupled with EPR data obtained on the ReNHase mutant enzymes showed subtle changes in the electronic environment around the active site Fe(III) ions, consistent with altering the hydrogen bonding interaction with the axial water ligand. X-ray crystal structures of the three PtNHase mutant enzymes confirmed the mutation and the lack of active site metal, while also providing insight into the active site hydrogen bonding network. Taken together, these data confirm that the conserved active site αSer residue plays an important catalytic role but is not essential for catalysis. They also confirm the necessity of the conserved second-sphere αSer residue for the metalation process and subsequent post-translational modification of the α-subunit in Co-type NHases but not Fe-type NHases, suggesting different mechanisms for the two types of NHases. SynopsisA strictly conserved active site αSer residue in both Co- and Fe-type nitrile hydratases was mutated. This αSer residue was found to play an important catalytic function, but is not essential. In Co-type NHases, it appears to be essential of active site maturation, but not for Fe-type NHases.

Nitrile hydratase as a promising biocatalyst: recent advances and future prospects.

Amides are an important type of synthetic intermediate used in the chemical, agrochemical, pharmaceutical, and nutraceutical industries. The traditional chemical process of converting nitriles into the corresponding amides is feasible but is restricted because of the harsh conditions required. In recent decades, nitrile hydratase (NHase, EC 4.2.1.84) has attracted considerable attention because of its application in nitrile transformation as a prominent biocatalyst. In this review, we provide a comprehensive survey of recent advances in NHase research in terms of natural distribution, enzyme screening, and molecular modification on the basis of its characteristics and catalytic mechanism. Additionally, industrial applications and recent significant biotechnology advances in NHase bioengineering and immobilization techniques are systematically summarized. Moreover, the current challenges and future perspectives for its further development in industrial applications for green chemistry were also discussed. This study contributes to the current state-of-the-art, providing important technical information for new NHase applications in manufacturing industries.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Structure of the Regulatory Region of Nitrile Hydratase Genes in Rhodococcus rhodochrous М8, a Biocatalyst for Production of Acrylic Heteropolymers

Structure of the Regulatory Region of Nitrile Hydratase Genes in Rhodococcus rhodochrous М8, a Biocatalyst for Production of Acrylic Heteropolymers

Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation.

Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

A Preblocking Strategy with Rapid Immobilization of Nitrile Hydratase and Precise Control of Hydrophobic Microenvironment for High Regioselective Amide Transformation

A Preblocking Strategy with Rapid Immobilization of Nitrile Hydratase and Precise Control of Hydrophobic Microenvironment for High Regioselective Amide Transformation

Biodegradation Potential of Cyanide and Nitrile using Bacteria of The Genus Rhodococcus

Cyanides and nitriles, characterized by their R-CN chains, are known for their toxicity, mutagenicity, and carcinogenicity, posing significant threats to environmental and human health. This study aims to explore the biodegradation capabilities of Rhodococcus sp. in breaking down cyanide and nitrile bonds. Rhodococcus pyridinivorans strain I-benzo was isolated from tanning waste and cultured in mineral media with a 20 mM benzonitrile substrate. The activity of this strain was tested using substrates such as benzonitrile, acetonitrile, acrylonitrile, benzamide, acetamide, and acrylamide, revealing positive reactions of nitrile hydratase and amidase enzymes through the Nessler measurement method, which indicated the production of ammonia and carboxylic acids. Furthermore, the degradation tests showed that the Vmax values for the biodegradation of potassium cyanide and sodium cyanide were 0.56 ppm/minute and 0.21 ppm/minute, respectively. These findings highlight the potential application of Rhodococcus pyridinivorans strain I-benzo in mitigating the environmental impact of cyanide and nitrile pollutants through efficient biodegradation.

A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

A CoII-Hydroxide Complex That Converts Directly to a CoII-Acetamide during Catalytic Nitrile Hydration.

In exploring structural and functional mimics of nitrile hydratases, we report the synthesis of the pseudo-trigonal bipyramidal CoII complexes (K)[CoII(DMF)(LPh)] (1(DMF)), (NMe4)2[CoII(OAc)(LPh)] (1(OAc)), and (NMe4)2[CoII(OH)(LPh)] (1(OH)) (LPh = 2,2',2''-nitrilo-tris-(N-phenylacetamide; DMF = N,N-dimethylformamide; -OAc = acetate)). The complexes were characterized using NMR, FT-IR, ESI-MS, electronic absorption spectroscopy, and X-ray crystallography, showing the LPh ligand to bind in a tetradentate tripodal fashion alongside the respective ancillary donor. One of the complexes, 1(OH), is an unusual structural and functional mimic of the Co active site in Co nitrile hydratases. 1(OH) reacted with acetonitrile to yield the CoII-acetamide complex (NMe4)2[CoII(NHC(O)CH3)(LPh)], 2, which was also thoroughly characterized. In the presence of excess hydroxide, 1(OH) was found to catalyze quantitative conversion of the added hydroxide into acetamide. Despite the differences in Co oxidation state in nitrile hydratases and 1(OH) (CoIII versus CoII, respectively), 1(OH) was nonetheless an effective nitrile hydration catalyst, selectively producing acetamide over multiple turnovers.

Design of new L-proline based terphenyl ligand: Binding interactions with cobalt (II) chloride and cobalt (III)-containing nitrile hydratase by spectroscopic and computational approach

A molecule having binding coordination with transition metal and metalloenzymes plays a crucial role in medicinal and industrial chemistry. Herein, we synthesize a new l-proline-based terphenyl ligand to investigate the binding interactions with cobalt (II) chloride and cobalt (III) containing nitrile hydratase by spectroscopic and theoretical approach. The formulated compound has been well characterized by elemental analysis, NMR (1H and 13C), FT-IR, Mass spectrometry. Moreover, the complete structure elucidation of synthesized l-proline-based terphenyl ligand was achieved via X-ray crystallography. The UV–visible spectroscopy and computational study reveals that the newly synthesized ligand confers a good binding response towards Cobalt (II) chloride and cobalt (III) containing nitrile hydratase enzyme as supported by calculated spectroscopic results and lower binding energy via molecular docking score, respectively. We hope that the present information can be utilized to develop new metal-coordinated compounds acting as useful metallodrugs or competitive artificial metalloenzymes.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Development of high-performance nitrile hydratase whole-cell catalyst by automated structure- and sequence-based design and mechanism insights

Development of high-performance nitrile hydratase whole-cell catalyst by automated structure- and sequence-based design and mechanism insights

Correction to: Purification and encapsulation of a nitrile hydratase (NHase) from Bacillus thuringiensis strain ZA96 isolated from oil‑contaminated soils

Correction to: Purification and encapsulation of a nitrile hydratase (NHase) from Bacillus thuringiensis strain ZA96 isolated from oil‑contaminated soils

Enhancing the catalytic activity of enzymes through tunnel amino acid modifications: The case of nitrile hydratase, a biocatalyst with posttranslational modifications used in amide production

Enhancing the catalytic activity of enzymes through tunnel amino acid modifications: The case of nitrile hydratase, a biocatalyst with posttranslational modifications used in amide production

Purification and encapsulation of a nitrile hydratase (NHase) from Bacillus thuringiensis strain ZA96 isolated from oil-contaminated soils

Purification and encapsulation of a nitrile hydratase (NHase) from Bacillus thuringiensis strain ZA96 isolated from oil-contaminated soils

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant βF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by βF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L−1 h−1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

An archaeal nitrile hydratase from the halophilic archaeon A07HB70 exhibits high tolerance to 3-cyanopyridine and nicotinamide

Nitrile hydratase (NHase, EC 4.2.1.84) is widely used in the industrial production of biosynthetic amide compounds. NHases obtained from prokaryotic and eukaryotic sources have been widely studied, while the NHases derived from archaeal sources have not been reported. Here, we focused on a distinctive NHase derived from a halophilic archaeon (archaeon A07HB70, A.r NHase) that thrives in high-salt environments. A notable feature of this enzyme is the natural fusion of the α subunit with the activator. A.r NHase retained 89.14 % of its activity after exposure to 4.0 M substrate and 97.52 % of its activity after exposure to 4.0 M product. These findings indicate that A.r NHase exhibits significantly higher tolerance to both substrate and product compared to NHases derived from other sources, which may be due to its unique genetic structure. The investigation of such highly stable archaeal NHase can offer a theoretical foundation for modifying NHase derived from other sources. This, in turn, would enhance the potential industrial application of NHase.

First insight into the natural biodegradation of cyanide in a gold tailings environment enriched in cyanide compounds

The disposal of mining wastes that contain cyanide are dumped in tailings from gold extraction is a public concern in mining countries such as South Africa. Many studies have shown the potential of microorganisms to degrade cyanide. However, no in-situ exploration, in tailings contaminated with cyanide, of the capability of indigenous microorganisms to act as a natural barrier for cyanide attenuation has been performed. Here we aim to combine geochemical and metagenomics techniques to reveal the genomic machinery of indigenous bacteria to degrade cyanide in gold tailings. Indigenous bacteria (i.e., Alicyclobacillus, Sulfobacillus, Acinetobacter, Achromobacter, and among others) pose the genomic machinery to trigger hydrolytic cyanide degradation using enzymes such as nitrilase, nitrile hydratase (Nhase) and thiocyanate. It seems that the lack of nitrogen sources induces the use of cyanide, which would contribute to its natural attenuation in the gold tailings. Therefore, the bacteria identified could represent the first barrier for the detoxification and degradation of cyanide in the outermost layer of the tailings.

General nitrogen regulation protein NtrC regulates the expression of nitrile hydratase and the synthesis of extracellular polysaccharide in Ensifer adhaerens CGMCC 6315

Ensifer adhaerens CGMCC 6315 contains two nitrile hydratases, CnhA and PnhA, which both convert the neonicotinoid insecticide acetamiprid to its amide metabolite. Here, we report that upregulation of PnhA and CnhA expression is related to nitrogen metabolism. Proteomic analysis showed that general nitrogen regulation protein NtrC was upregulated under nutrient deprivation. We thus constructed a ΔntrC mutant strain of E. adhaerens CGMCC 6315. In the ΔntrC strain, when cells were cultured in minimal salts medium supplemented with ammonium chloride, acetamiprid degradation and the expression of cnhA and pnhA were enhanced relative to those in wild-type E. adhaerens CGMCC 6315. In addition, NtrC negatively regulates synthesis of extracellular polysaccharides in this bacterium. The germination rate of soybean seeds was improved by inoculation with the ΔntrC strain of E. adhaerens CGMCC 6315 compared with the wild-type. This study is the first to report regulation of the expression of nitrile hydratase by NtrC, which is important for the practical remediation of pesticide pollution.

Residues of sulfoxaflor and its metabolites in floral and extrafloral nectar from Hibiscus rosa-sinensis L. (Malvaceae) with or without co-application of tebuconazole

Systemic pesticide exposure through nectar is a growing global concern linked to loss of insect diversity, especially pollinators. The insecticide sulfoxaflor and the fungicide tebuconazole are currently widely used systemic pesticides which are toxic to certain pollinators. However, their metabolisms in floral or extrafloral nectar under different application methods have not yet been well studied. Hibiscus rosa-sinensis was exposed to sulfoxaflor and tebuconazole via soil drenching and foliar spraying. Sulfoxaflor, tebuconazole, and their main metabolites in floral and extrafloral nectar, soil, and leaves were identified and quantified using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ MS). The chemical compositions of unexposed and contaminated H. rosa-sinensis floral nectar or extrafloral nectar were compared using regular biochemical methods. The activities of two pesticide detoxifying enzymes, glutathione-s-transferase and nitrile hydratase, in H. rosa-sinensis nectar were examined using LC-MS and spectrophotometry. The floral nectar proteome of H. rosa-sinensis was analysed using high-resolution orbitrap-based MS/MS analysis to screen for sulfoxaflor and tebuconazole detoxifying enzymes. H. rosa-sinensis can absorb sulfoxaflor and tebuconazole through its roots or leaf surfaces and secrete them into floral nectar and extrafloral nectar. Both sulfoxaflor and tebuconazole and their major metabolites were present at higher concentrations in extrafloral nectar than in floral nectar. X11719474 was the dominant metabolite of sulfoxaflor in the nectars we studied. Compared with soil application, more sulfoxaflor and tebuconazole remained in their original forms in floral nectar and extrafloral nectar after foliar application. Sulfoxaflor and tebuconazole exposure did not modify the chemical composition of floral or extrafloral nectar. No active components, including proteins in the nectar, were detected to be able to detoxify sulfoxaflor.