Catalytic mechanism of nitrile hydratase proposed by time-resolved X-ray crystallography using a novel substrate, tert-butylisonitrile

Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteine-sulfinic 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 reflectance-Fourier 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 alphaCys114. These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by alphaCys114-SOH.     

Post-translational modification is essential for catalytic activity of nitrile hydratase

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.     

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.     

High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism

The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3A resolution. The two cysteine residues (alphaCys(112) and alphaCys(114)) equatorially coordinated to the ferric ion were post-translationally modified to cysteine sulfinic acids. A glutamine residue (alphaGln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion.     

Fe-type nitrile hydratase

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.     

Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt-containing NHase (Co-NHase) was reported to depend on self-subunit swapping and the activator of the Co-NHase acts as a self-subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit-fused Co-NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL-18668 and the activator of low-molecular-mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt-containing proteins. Substitution of the amino acids involved in the C-terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt-binding sites might be located at the flexible C-terminal region. The cobalt-free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit-fused NHases and confirmed the metallochaperone function of the self-subunit swapping chaperone.     

Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy

Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.     

Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center

Arginine 56 in the β subunit (βArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (–SO₂H) and sulfenyl (–SOH) groups of the post-translationally modified cysteine residues in the catalytic center. βArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The βR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the βR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV–visible absorption and light-induced Fourier transform infrared difference spectra suggest that βArg56 is involved in the positioning of the –SO₂H and –SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, βArg56 is essential for catalysis.     

Molecular dynamics simulations of the photoactive protein nitrile hydratase

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, α and β. Hydrogen bonds between βArg-56 and αCys-114 sulfenic acid (αCEA114) 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 λ < 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.     

Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined. Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases. In particular, the Phe52 in the beta subunit of NHase from B. smithii covers the metal center partially like a small lid and narrows the active site cleft. It is well known that the NHase from B. smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme. This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.     

腈类物降解菌多样性和产腈水合酶研究进展

腈水合酶催化反应在有机合成领域已有广泛的应用。作为一类重要的催化剂,腈水合酶可以将腈类物质转化为相应的酰胺。由于这种酶具有固有的立体和区域选择性,在精细化工领域已成为绿色、温和、对同分异构体具有选择性的催化剂。同时腈水合酶在生物修复和环境保护中也起着重要作用。综述了目前国内外腈水合酶的研究进展,包括降解腈类的微生物多样性、腈水合酶的催化特性、产腈水合酶菌株的改造以及腈水合酶相关基因的克隆与研究。对固定化酶和腈水合酶的应用也进行了叙述。     

A Nitrile Hydratase in the Eukaryote Monosiga brevicollis

Bacterial nitrile hydratase (NHases) are important industrial catalysts and waste water remediation tools. In a global computational screening of conventional and metagenomic sequence data for NHases, we detected the two usually separated NHase subunits fused in one protein of the choanoflagellate Monosiga brevicollis, a recently sequenced unicellular model organism from the closest sister group of Metazoa. This is the first time that an NHase is found in eukaryotes and the first time it is observed as a fusion protein. The presence of an intron, subunit fusion and expressed sequence tags covering parts of the gene exclude contamination and suggest a functional gene. Phylogenetic analyses and genomic context imply a probable ancient horizontal gene transfer (HGT) from proteobacteria. The newly discovered NHase might open biotechnological routes due to its unconventional structure, its new type of host and its apparent integration into eukaryotic protein networks.     

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.     

Improvement of stability of nitrile hydratase via protein fragment swapping

Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase fragment from Comamonas testosteroni 5-MGAM-4D and two fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of PpNHase. Seven chimeric NHases were designed using STAR (site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. All constructed chimeric NHases showed 1.4- to 3.5-fold enhancement in thermostability and six of them become more tolerant to high-concentration product. Notably, one of these NHases, 3AB, exhibited a 1.4 ± 0.05-fold increase in activity compared to the wild-type PpNHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type PpNHase. The 3AB constructed in this study is useful for further industrial application, and the method for designing the chimeric protein using homologous protein fragment swapping without a decrease in activity may be a strategy to improve the stability of other enzymes.     

New inhibitors of iron-containing nitrile hydratases

There is growing evidence in the literature emphasizing the significance of the post-translational modification of cysteine thiols to sulfenic acids (SOH), which have been found in a number of proteins. Crystallographic and mass spectrometric evidence has shown the presence of this group in an inactive form of the industrially important enzyme nitrile hydratase (NHase). This oxidized cysteine is unique in that it forms part of the coordination sphere of the low-spin iron III at the active site of the enzyme. The presence of this unstable sulfenic group in the active form of NHase is the subject of some controversy. To try to detect this function in NHase, we have studied the inhibitory effect on nitrile hydration of reagents known to react with sulfenic acids. Two NHases were studied, namely, Rhodococcus rhodochrous R312 NHase and Comamonas testosteroni NI1 NHase, and the reagents used were meta-chlorocarbonyldicyano-phenylhydrazone (m-ClCP), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), and 2-nitro-5-thiocyanato-benzoic acid (NTBA). Following this approach we report three novel inhibitors of NHases. In addition, we report thiocyanate reagents that can be used to monitor NHase activity spectroscopically.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

Cobalt-substituted Fe-type nitrile hydratase of Rhodococcus sp. N-771

When the genes encoding alpha and beta subunits of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 were expressed in Escherichia coli in Co-supplemented medium without co-expression of the NHase activator, the NHase specifically incorporated not Fe but Co ion into the catalytic center. The produced Co-substituted enzyme exhibited rather weak NHase activity, initially. However, the activity gradually increased by the incubation with an oxidizing agent, potassium hexacyanoferrate. The oxidizing agent is likely to activate the Co-substituent by oxidizing the Co atom to a low-spin Co(3+) state and/or modification of alphaCys-112 to a cysteine-sulfinic acid. It is suggested that the NHase activator not only supports the insertion of an Fe ion into the NHase protein but also activates the enzyme via the oxidation of its iron center.