Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase

Three cysteine residues, which are completely conserved among alpha-subunits in all nitrile hydratases, are thought to be the ligands of a metal ion in the catalytic center of this enzyme. These cysteine residues (i.e. alpha C102, alpha C105 and alpha C107) in the high-molecular-mass nitrile hydratase (H-NHase) of Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using the R. rhodochrous ATCC12674 host-vector system, and the resultant transformants were investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the cell-free extracts of each mutant transformant revealed that four mutant transformants (i.e. alpha C105A, alpha C107A, alpha C102A/C105A and alpha C105A/C107A) showed predominant alpha- and beta-subunit protein bands with a mobility identical to those of the native H-NHase, while three mutant transformants (i.e. alpha C102A, alpha C102A/C107A and alpha C102A/C105A/C107A) did not produce the corresponding proteins. The purified former four mutant enzymes showed neither enzymatic activity nor the maximum absorption at 410 nm which was detected in the wild type H-NHase. They also did not contain cobalt ions. Based upon these findings, these three cysteine residues were found to be essential for the active expression of H-NHase.     

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.     

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.     

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.     

An over expression and high efficient mutation system of a cobalt-containing nitrile hydratase

A superior novel recombinant strain, E. coli BL21(DE3)/pETNH^M, containing the start codon mutation of the subunit, was constructed and selected as an overexpression and high efficient mutation platform for the genetic manipulation of the nitrile hydratase (NHase). Under optimal conditions, the specific activity of the recombinant strain reached as high as 452 U/mg dry cell. Enzymatic characteristics studies showed that the reaction activation energy of the recombinant NHase^M was 24.4 ± 0.5 kJ/mol, the suited pH range for catalysis was 5.5–7.5, and the K_m value was 4.34 g/L (82 mM). To assess the feasibility of the NHase improvement by protein rational design using this E. coli, site-directed mutagenesis of S122A, S122C, S122D and βW47E of the NHase^M were carried out. The NHase^M (S122A) and NHase^M (S122D) mutants were entirely inactive due to the charge change of the side-chain group. The product tolerance of the NHase^M (S122C) mutant was enhanced while its activity decreased by 30%. The thermo-stability of the NHase^M (βW47E) mutant was significantly strengthened, while its activity reduced by nearly 50%. These results confirmed that the specific activity of the mutant NHase expressed by the recombinant E. coli BL21(DE3)/pETNH^M can reasonably change with and without mutations. Therefore, this recombinant E. coli can be efficiently and confidently used for the further rational/random evolution of the NHase to simultaneously improve the activity, thermo-stability and product tolerance of the target NHase.     

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     

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.     

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.     

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.     

A novel thermostable nitrile hydratase

A novel, nitrile-degrading, thermophilic microorganism belonging to the genus Bacillus and most closely related to strain DSM 2349 has been isolated. The strain grew optimally at 65°C with the constitutive expression of a thermostable intracellular nitrile hydratase. No aromatic-specific "benzonitrilase" activity was detected under any conditions. The enzyme, an α₂β₂ heterotetramer with a native molecular weight of 110 kDa, was purified to homogeneity. N-terminal sequence data showed no homology to known bacterial α subunit sequences but had a high level of identity with other bacterial N-terminal β subunit sequences. The purified enzyme had a broad pH-activity range (50% activity limits were pH 5.1 and 8.7) and was stable in aqueous solution up to 60°C in the absence of either substrates or substrate analogues. Substrate specificity was restricted to aliphatic nitriles, but an unusual preference for branched and cyclic aliphatic nitriles was noted. Turnover rates under optimum reaction conditions were 746 and 4580 sec⁻¹ for acetonitrile and valeronitrile, respectively. Received: December 1, 1997 / Accepted: February 24, 1998     

Purification of inactivated photoresponsive nitrile hydratase

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35 degrees C, and lost its activity above 40 degrees C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30 degrees C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed form after photoirradiation.     

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.     

对羟基苯乙腈水合酶高产菌株的选育

从全省各处采集的50多份土样筛选到一株产腈水合酶能力较高的菌株E10a,该菌株产生的腈水合酶为非诱导酶,产酶条件优化实验结果表明,产酶培养基组成:麦芽糖20 g/L,酵母膏5 g/L,尿素7.5 g/L,味精0.75 g/L,K2HPO40.5 g/L,KH2PO40.5 g/L,MgSO40.5 g/L,FeSO4.7H2O 10 mg/L,CoCl210 mg/L,微量元素母液0.8 ml/L;最佳培养条件为:培养温度28℃,摇床转速150 r/min,培养基起始pH 7.0,培养时间5 d,在优化培养条件下,1 h可将1 g/L质量浓度的底物对羟基苯乙腈全部转化为对羟基苯乙酰胺。     

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the -substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted -hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of -hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.     

Molecular characterisation of a novel thermophilic nitrile hydratase

The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases.     

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。     

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.     

产腈水合酶菌Nocardia sp.HD9611的发酵条件优化的研究

探讨了种龄、接种量、搅拌转速、pH及补料等因素对Nocardia sp.HD9611产腈水合酶的影响.结果表明,最佳种龄为20h;接种量对酶活的提高没有明显影响,但7.5%时最佳;当搅拌转速低于400r/min时,溶解氧将成为细胞生长的限制因子;发酵过程中pH调节对细胞量及酶活的提高有积极的作用;补料对细胞密度及酶的产生有积极影响,总糖为80g/L时,细胞量31.88g/L,提高了120.8%,酶活为7100U,提高了107.6%.此研究为制定最佳控制策略提供了参考.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

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.