Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009

A study has been carried out into the biocatalytic hydration of nitriles using the nitrile hydratase enzyme from Rhodopseudomonas palustris CGA009. It has been shown that this nitrile hydratase can hydrate aliphatic, aromatic and heterocyclic nitriles under very mild conditions, in mixtures of pH 7 buffer and a range of organic solvents, often with excellent chemoselectivity. The major determinant of hydration occurring is the degree of steric hindrance around the nitrile moiety and/or size of the substrates.     

腈水合酶的发酵及酶学性质

红球菌Rhodococceus rhodochrous J1在含有脲和钴的培养基中发酵产生腈水合酶,可以催化丙烯腈水合为丙烯酰胺。调节培养条件使发酵液的酶活力达到6150u／ml。温度30℃,反应体系呈中性酶活性很高并且稳定。底物浓度,酶浓度,反应时间的正交实验表明,底物丙烯腈是显著因子,最佳浓度7％,超过此浓度酶会失活;1min内,水合反应转化率可达80％。浓度超过15％的丙烯酰胺会抑制酶的活性。     

Bioconversion of acrylonitruke to acrylamide using a thermostable nitrile hydratase

Although providing an attractive route for production of crrylamide from acrylonitrile, utilization of nitrile hydratase en zymes has been limited by the requirement for low temperatu rebioconversion conditions. Thisrreportsummarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrileatt concentrations to 1% at elevated temperatures. A new isolate Bacillus sp. BR449 constitutively expresses a thermostble nitrile hydratase with properties iccluding low substrate inhibition and broad temperature range with optimal activity at 55°C. With prolonged exposure, BR449 nitrile hydratase exhibited temperature-dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzymes/     

Oxygenation of thiolates to S-bonded sulfinate in an iron III complex related to nitrile hydratase

Air oxidation of the iron(III) complex derived from (2-mercaptoisobutyryl)-4,5-dichloro-O-phenylenediamine in the presence of Et4NCN afforded the corresponding disulfinato species. With two carboxamido nitrogens trans to two S-bonded sulfinates, this complex mimics the disulfinate inactive form of Fe-NHase.     

Theoretical investigation of the first-shell mechanism of nitrile hydratase

The first-shell mechanism of nitrile hydratase (NHase) is investigated theoretically using density functional theory. NHases catalyze the conversion of nitriles to amides and are classified into two groups, the non-heme Fe(III) NHases and the non-corrinoid Co(III) NHases. The active site of the non-heme iron NHase comprises a low-spin iron (S=1/2) with a remarkable set of ligands, including two deprotonated backbone nitrogens and both cysteine-sulfenic and cysteine-sulfinic acids. A widely proposed reaction mechanism of NHase is the first-shell mechanism in which the nitrile substrate binds directly to the low-spin iron in the sixth coordination site. We have used quantum chemical models of the NHase active site to investigate this mechanism. We present potential energy profiles for the reaction and provide characterization of the intermediates and transition-state structures for the NHase-mediated conversion of acetonitrile. The results indicate that the first-shell ligand Cys114-SO- could be a possible base in the nitrile hydration mechanism, abstracting a proton from the nucleophilic water molecule. The generally suggested role of the Fe(III) center as a Lewis acid, activating the substrate toward nucleophilic attack, is shown to be unlikely. Instead, the metal is suggested to provide electrostatic stabilization to the anionic imidate intermediate, thereby lowering the reaction barrier.     

The role of post-translational modification in the photoregulation of Fe-type nitrile hydratase

The inactive, nitrosyl bound form of Fe-type nitrile hydratase (NHase) contains two active site cysteine residues that are post-translationally modified to sulfenate (SO-) and sulfinate (SO2-) ligands. DFT and INDO/S calculations support the hypothesis that these unusual modifications play a key role in modulating the electronic absorption spectra and photoreactivity of the Fe(III) centre in the enzyme.     

Formation and purification of nitrile hydratase fromCorynebacterium pseudodiphteriticum ZBB-41

A strain of nitrile hydratase-forming microorganism,Corynebacterium pseudodiphteriticum ZBB-41, was isolated from soil and the conditions for the enzyme formation have been studied. Addition of ferric or ferrous ions and the use of methacrylamide as an inducer greatly enhanced enzyme formation. When the strain was cultivated at 27°C for 64 h in an optimum medium, the specific activity of the enzyme was 83U/mg of dry cell weight. The enzyme was purified from the crude extract of methacrylamide induced cells in 6 steps with 9.5-fold purification and 8.8% recovery. The enzyme has a molecular mass of 80,000 and consists of two subunits with molecular mass of 28,000 and 25,000 respectively. The isoelectric point was 4.2. The Km value to acrylonitrile was 0.21 M. The enzyme was strongly inhibited by Hg²⁺, Cu²⁺, Ag⁺, iodoacetate, and phenyl mercuric acetate and can be protected by 2-mercaptoethanol. The enzyme was noncompetitively inhibited by sodium cyanide, the Ki value was 0.13 mM.     

Probing the influence of local coordination environment on the properties of Fe-type nitrile hydratase model complexes

A series of four structurally related cis-dithiolate-ligated Fe(III) complexes, [Fe(III)(DITpy)2]Cl (1), [Fe(III)(DITIm)2]Cl (2), [Fe(III)(ADIT)2]Cl (3), and [Fe(III)(AMIT)2]Cl (4), are described. The structural characterization of 3 as well as the spectroscopic properties of 3 and 4 has been previously reported. Crystal data for 1, 2, and 4 are as follows: 1.3H2O crystallizes in the orthorhombic space group Pca2(1) with a = 19.800(4) A, b = 18.450(4) A, c = 14.800(3) A, and Z = 8. 2.(1/2)EtOH.1/2H2O crystallizes in the monoclinic space group Cc with a = 24.792(4) A, b = 14.364(3) A, c = 17.527(3) A, beta = 124.91(2) degrees, and Z = 8. 4 crystallizes in the triclinic space group P1 with a = 8.0152(6) A, b = 10.0221(8) A, c = 11.8384(10) A, alpha = 73.460(3) degrees, beta = 71.451(5) degrees, gamma = 72.856(4) degrees, and Z = 2. Complexes 1-4 share a common S2N4 coordination environment that consists of two cis-thiolates, two trans-imines, and two cis-terminal nitrogen donors: Nterm = pyridine (1), imidazole (2), and primary amine (3 and 4). The crystallographically determined mean Fe-S bond distances in 1-4 range from 2.196 to 2.232 A and are characteristic of low-spin Fe(III)-thiolate complexes. The low-spin S = 1/2 ground state was confirmed by both EPR and magnetic susceptibility measurements. The electronic spectra of these complexes are characterized by broad absorption bands centered near approximately 700 nm that are consistent with ligand-to-metal charge-transfer (CT) bands. The complexes were further characterized by cyclic voltammetry measurements, and all possess highly negative Fe(III)/Fe(II) redox couples ( approximately -1 V vs SCE, saturated calomel electrode) indicating that alkyl thiolate donors are effective at stabilizing Fe(III) centers. Both the redox couple and the 700 nm band in the visible spectra show solvent-dependent shifts that are dependent upon the H-bonding ability of the solvent. The implications of these results with respect to the active site of the iron-containing nitrile hydratases are also discussed.     

The first example of a monomeric alumatrane

The novel five-coordinate aluminum adduct Me(2)HN. AlL (2) [wherein L = tris(2-oxy-3,5-dimethylbenzyl)amine] containing three six-membered rings has been characterized by spectroscopic and by X-ray means. This adduct of an alumatrane is the first structurally characterized monomeric alumatrane derivative, and unlike its parent alumatrane [Al(OCH(2)CH(2))(3)N](x), 2 is monomeric in the gas, solution, and solid states. The X-ray molecular structure of 2 reveals a tricyclic cage moiety of C(3) symmetry. The aluminum geometry is a slightly distorted trigonal bipyramid in which, quite unexpectedly, the metal atom is located somewhat below the plane formed by three equatorial oxygens and its Al-N(tertiary) bond is shorter than that in Me(3)N.AlH(3).NMe(3) (Heitsch, C. W.; Nordman, C. E.; Parry, R. W. Inorg. Chem. 1963, 2, 508).     

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.     

Activation of nitriles by hydrogen bonding in cycloadditions with nitrile oxides

The dipolarophilic activity of aromatic nitriles in cycloaddition with benzonitrile oxides is remarkably enhanced by ortho-acylamino substituents. The activation depends upon the solvent and can be ascribed to a hydrogen bond which assists cycloaddition.     

C-Silylation de composes a fonction nitrile. Synthese de nitriles α-silicies

The direct silylation by Me₃SiCl/Li/THF of saturated nitriles having a hydrogen atom in the α position with respect to the function leads, according to a simple and rapid process, to the corresponding α-silylated nitriles in yields that are moderate but much higher than those given by the comparable previously known routes. α, β-Unsaturated nitriles give the derivatives resulting from the disilylation of the double bond. Allyl or benzyl cyanide and diphenyl acetonitrile first undergo the elimination of the nitrile group (which behaves as a halogen atom) whereas cyanamid affords bis(trimethylsilyl)carbodiimide giving after further silylation the unexpected tris(trimethylsilyl)amine.     

A powerful new nitrile hydratase for organic synthesis — aromatic and heteroaromatic nitrile hydrolyses — a rationalisation

A powerful new nitrile hydratase organism, Rhodococcus rhodocrous AJ270 has been isolated that efficiently hydrolyses all kinds of nitriles to amides and/or acids. This paper shows that aromatic and heterocyclic nitriles are readily hydrolysed to acids but, that those bearing an adjacent-substituent (which may be an ortho substituent or an adjacent heteroatom in the ring) give amides in good yield but only slowly proceed to acids.     

The first example of a copper(II) chloride complex with 1,3-thiazolidine-2-thione

Summary The reaction of copper(II) chloride with 1,3-thiazolidine-2-thione in absolute ethanol yields the complex CuL₂Cl₂. This derivative has been studied by elemental analyses, molar conductance, magnetic moment measurements at different temperatures, electronic and infrared spectroscopy, thermal analyses (TG and DTG) and e.p.r. techniques. The complex has a pseudooctahedral stereochemistry, with bridging halides and with the ligands monodentate-S-bondedvia the exocyclic sulphur atom.     

Influence of sequential thiolate oxidation on a nitrile hydratase mimic probed by multiedge X-ray absorption spectroscopy

Nitrile hydratases (NHases) are Fe(III)- and Co(III)-containing hydrolytic enzymes that convert nitriles into amides. The metal-center is contained within an N(2)S(3) coordination motif with two post-translationally modified cysteinates contained in a cis arrangement, which have been converted into a sulfinate (R-SO(2)(-)) and a sulfenate (R-SO(-)) group. Herein, we utilize Ru L-edge and ligand (N-, S-, and P-) K-edge X-ray absorption spectroscopies to probe the influence that these modifications have on the electronic structure of a series of sequentially oxidized thiolate-coordinated Ru(II) complexes ((bmmp-TASN)RuPPh(3), (bmmp-O(2)-TASN)RuPPh(3), and (bmmp-O(3)-TASN)RuPPh(3)). Included is the use of N K-edge spectroscopy, which was used for the first time to extract N-metal covalency parameters. We find that upon oxygenation of the bis-thiolate compound (bmmp-TASN)RuPPh(3) to the sulfenato species (bmmp-O(2)-TASN)RuPPh(3) and then to the mixed sulfenato/sulfinato speices (bmmp-O(3)-TASN)RuPPh(3) the complexes become progressively more ionic, and hence the Ru(II) center becomes a harder Lewis acid. These findings are reinforced by hybrid DFT calculations (B(38HF)P86) using a large quadruple-ζ basis set. The biological implications of these findings in relation to the NHase catalytic cycle are discussed in terms of the creation of a harder Lewis acid, which aids in nitrile hydrolysis.     

The first structurally characterized nitrosyl heme thiolate model complex

The NO ligand in the formally {FeNO}6 compound [Fe(oep)(NO)(thiolate)] is bent, and does not impart a significant structural trans effect to the Fe-S bond.     

A comparative DFT study of substrates and products of industrial enzyme nitrile hydratase

Nitrile hydratase (NHase) is a metalloenzyme used in industrial biotechnology for a large scale production of common chemicals. NHases convert nitriles to the corresponding amides. Although the structures of some forms of NHases containing nonheme low spin Fe(III) or low spin noncorrinoid Co(III) are known, neither a catalytic mechanism nor the reasons of high selectivity towards aromatic ligands are recognized. Optimized geometries, molecular electrostatic potential maps and infrared spectra of commercially important aromatic substrates of the NHase (nicotinonitrile, o‐, m‐, p‐methylbenzonitrile) and the corresponding products (nicotinamide, o‐, m‐, p‐methylbenzamid) were investigated using the density functional theory method with the B3LYP functional and the 6‐31G(d,p) basis set. Calculated hypothetical intrinsic reaction paths indicate that benzimidic acids may be involved as intermediates. This study elucidates differences in the electronic properties of substrates and products of NHases, provides an insight into the molecular basis of the catalytic reaction and helps to explain varying enzymatic activities of microbial NHases. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

The first example of electrophilic substitution on a benzannulene

Electrophilic substitution (nitration, acylation) proceeds readily in the macro ring of the benzannelated [14]annulene 1 to give products similar to that of the non annulated parent 6.     

Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli

Expression of nitrile hydratase enzymes utilized in a new “green” process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobaltion into this host. We describe the expression of a thermostable nitrile hydratase from a moderatethermophile Bacillus sp. BR449 in E. coli in which the cobaltrequired for enzyme activation is introduced by incubation, of the apoenzyme in the presence of Co⁺⁺ ion at 50°C, yielding active and thermostable, enzyme.