Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1

The formation of nitrile hydratase required cobalt ions in Rhodococcus rhodochrous J1. No other transition-metals could replace the cobalt ion. The Rhodococcus nitrile hydratase was purified to homogeneity and found to contain a cobalt atom. The occurrence of a cobalt-induced and cobalt-containing nitrile hydratase, different from the nitrile hydratases in Pseudomonas chlororaphis B23 and Brevibacterium R312 containing a ferric ion in their active center, has been demonstrated here for the first time.     

Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Crystal structure of cobalt-containing nitrile hydratase.

The crystal structure of cobalt-containing nitrile hydratase from Pseudonocardia thermophila JCM 3095 at 1.8 A resolution revealed the structure of the noncorrin cobalt at the catalytic center. Two cysteine residues (alphaCys(111) and alphaCys(113)) coordinated to the cobalt were posttranslationally modified to cysteine-sulfinic acid and to cysteine-sulfenic acid, respectively, like in iron-containing nitrile hydratase. A tryptophan residue (betaTrp(72)), which may be involved in substrate binding, replaced the tyrosine residue of iron-containing nitrile hydratase. The difference seems to be responsible for the preference for aromatic nitriles rather than aliphatic ones of cobalt-containing nitrile hydratase.     

Fed-batch fermentation for production of nitrile hydratase byRhodococcus rhodochrous M33

To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Influence of cobalt substitution on the activity of iron-type nitrile hydratase: are cobalt type nitrile hydratases regulated by carbon monoxide?

Comamonas testosteroni Ni1 nitrile hydratase is a Fe-type nitrile hydratase whose native and recombinant forms are identical. Here, the iron of Ni1 nitrile hydratase was replaced by cobalt using a chaperone based Escherichia coli expression system. Cobalt (CoNi1) and iron (FeNi1) enzymes share identical Vmax (30 nmol min(-1) mg(-1)) and Km (200 microM) toward their substrate and identical Ki values for the known competitive inhibitors of FeNi1. However, nitrophenols used as inhibitors do display a different inhibition pattern on both enzymes. Furthermore, CoNi1 and FeNi1 are also different in their sensitivity to nitric oxide and carbon monoxide, CO being selective of the cobalt enzyme. These differences are rationalized in relation to the nature of the catalytic metal center in the enzyme.     

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

Summary We sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.Offprint requests to: T. Nagasawa     

Expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in Pichia pastoris

The nitrile hydratase (NHase) gene of Rhodococcus rhodochrous PA-34 mutant 4D has been amplified by PCR, cloned and expressed in Pichia pastoris KM-71 using pHIL-D2 expression vector. The recombinant P. pastoris KM-71 exhibited active expression of the nitrile hydratase gene of the mutant 4D and has shown very good potential for the transformation of 3-cyanopyridine to nicotinamide. The recombinant P. pastoris KM-71 exhibited maximum NHase activity when cultivated in YPD medium was supplemented with 0.4?mM cobalt ions. The recombinant P. pastoris KM-71 showed maximum nitrile hydratase enzyme production, when incubated at 30?°C for 15?h.     

Nitrile hydratase from a thermophilic Bacillus smithii

A novel thermophilic Bacillus smithii strain SC-J05-1, isolated from a hot spring, had the ability of hydrating nitrile to form amide. The nitrile hydratase was purified to homogeneity from the microbial cells of SC-J05-1 and was characterized. The enzyme was a 130-kDa protein composed of two different subunits (25.3 kDa and 26.8 kDa) and contained cobalt ions. This enzyme had the optimal temperature of 40°C and was stable up to 50°C. The optimal pH was in the alkaline region higher than pH 10. Received 02 September 1997/ Accepted in revised form 06 February 1998     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced.     

Characterisation of the nitrile hydratase gene clusters of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strains AJ270 and AJ300 and <Emphasis Type="Italic">Microbacterium</Emphasis> sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.     

Nitrile hydratase genes are present in multiple eukaryotic supergroups

Background

Nitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.

Methodology/Principal Findings

Here we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.

Conclusions/Significance

The evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.     

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.     

一类生物催化剂——氰基耐受型腈水合酶

氰基耐受型腈水合酶是一类生物催化剂。与普通腈水合酶相比,它能够耐受体系中较高浓度的氰基而不受抑制,从而为α-羟(氨)基酰胺的工业化合成开辟了崭新途径。研究腈水合酶的氰基耐受性机理及提高其耐受能力是目前需要解决的关键问题。综述了腈水合酶受氰基抑制的机制,氰基耐受型腈水合酶的发现以及其在蛋氨酸和2-羟基异丁酰胺生物合成中的应用。同时,对今后氰基耐受型腈水合酶基础、应用研究的思路进行了探讨。     

A study of the catalytic properties of the nitrile hydratase immobilized on aluminum oxides and carbon-containing adsorbents

The nitrile hydratase isolated from Rhodococcus ruber strain gt1, displaying a high nitrile hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon support Sibunit. The activity and operational stability of the immobilized nitrile hydratase were studied in the reaction of acrylonitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the support led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The nitrile hydratase immobilized on Sibunit and carbon-containing aluminum α-oxide having a “crust” structure displayed the highest operational stability in acrylonitrile hydration. It was shown that the thermostability of adsorbed nitrile hydratase increased by one order of magnitude.     

Iron and cobalt complexes of 2,6-diacetylpyridine-bis(R-thiosemicarbazone) (R = H, phenyl) showing unprecedented ligand deviation from planarity

The syntheses, characterization, and single-crystal X-ray crystal structures are reported for four complexes of iron and cobalt with the pentadentate ligands, 2,6-diacetylpyridinebis(thiosemicarbazone) (H₂L¹) and 2,6-diacetylpyridinebis(phenylthiosemicarbazone) (H₂L²), including a cobalt dimer displaying a deviation from planarity which is unprecedented for this class of ligands and allows the ligand to occupy five positions of a pseudo-octahedral coordination sphere. This dimer reacts with KCN to produce a mononuclear complex of relevance to the active site of cobalt nitrile hydratase.     

Pathological consequences of copper deficiency and cobalt deficiency

Aspects of the pathology of copper deficiency in several species, and cobalt deficiency in sheep, are summarized. An attempt is made to interpret morphological changes in copper-deficient animals in terms of biochemical defects. The common denominator may be mitochondrial lesions, with a generalized effect on energy-dependent synthetic functions of the cell. In copper deficiency, such defects can be attributed to depletion of copper-dependent enzymes, while deficiency of cobalt in ruminants is, in effect, deficiency of vitamin B12. The pathological consequences of vitamin B12 deficiency form a syndrome, notable features of which are neurological and muscular lesions, in which the metabolic consequences of hepatic damage may play a significant role.     

Effect of porphyrins and their derivatives on biosynthesis of vitamin B 12 and the development of Propionibacterium shermanii]

The effect of uroporphyrin, coproporphyrin and their cobalt-containing derivatives on the biosynthesis of vitamin B12 and development of propionibacterium shermanii was studied. The compounds under study stimulated the vitamin synthesis by growing cultures and resting suspensions of these bacteria. Cobalt porphyrins as the sole source of cobalt were used in the vitamin B12 biosynthesis. An addition of cobalt porphyrins to the growing culture of propionic bacteria increased in accumulation of their biomass. Possible mechanisms of porphyrin involvement in the biosynthesis of vitamin B12 and the specific role of cobalt porphyrins in the bacterial activity are discussed.     

Catalytic properties of a nitrile hydratase immobilized on activated chitosan

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V _max), whereas the Michaelis constant (K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.