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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, alpha and beta. Hydrogen bonds between betaArg-56 and alphaCys-114 sulfenic acid (alphaCEA114) 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 lambda < 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.

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Enzymes are essential for the catalysis of biochemical reactions and in the regulation of metabolic pathways. They function by greatly accelerating the rate of specific chemical reactions that would otherwise be slow. It has been shown that extremely low-power microwaves can influence enzyme activity [1-5]. This study is focused at investigating the effects of low level microwave exposures ranging from 500MHz to 900MHz on L-Lactate Dehydrogenase (LDH) enzyme activity. The results obtained revealed the increased bioactivity of the LDH upon microwave radiation at two particular frequencies 500MHz and 900MHz.

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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.

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The alphabeta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH(-). The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NH(dark). The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NH(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with Mössbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(light) with the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mössbauer spectroscopy. Photolysis at 4.2 K in the Mössbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.

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BACKGROUND: Nitrile hydratases are unusual metalloenzymes that catalyze the hydration of nitriles to their corresponding amides. They are used as biocatalysts in acrylamide production, one of the few commercial scale bioprocesses, as well as in environmental remediation for the removal of nitriles from waste streams. Nitrile hydratases are composed of two subunits, alpha and beta, and they contain one iron atom per alphabeta unit. We have determined the crystal structure of photoactivated iron-containing nitrile hydratase from Rhodococcus sp. R312 to 2.65 A resolution as a first step in the elucidation of its catalytic mechanism. RESULTS: The alpha subunit consists of a long N-terminal arm and a C-terminal domain that forms a novel fold. This fold can be described as a four layered structure, alpha-beta-beta-alpha, with unusual connectivities between the beta strands. The beta subunit also contains a long N-terminal extension, a helical domain, and a C-terminal domain that folds into a beta roll. The two subunits form a tight heterodimer that is the functional unit of the enzyme. The active site is located in a cavity at the subunit-subunit interface. The iron centre is formed by residues from the alpha subunit only-three cysteine thiolates and two mainchain amide nitrogen atoms are ligands. CONCLUSIONS: Nitrile hydratases contain a novel iron centre with a structure not previously observed in proteins; it resembles a hybrid of the iron centres of heme and Fe-S proteins. The low-spin electronic configuration presumably results in part from two Fe-amide nitrogen bonds. The structure is consistent with the metal ion having a role as a Lewis acid in the catalytic reaction.

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Nitrile hydratase (NHase) from Rhodococcus sp. N-771, which contains a non-heme iron center in the catalytic site, has been known to be activated by light illumination. Recently, endogenous nitric oxide (NO) was found in this enzyme by FTIR spectroscopy [Noguchi et al. (1995) FEBS Lett. 358, 9-12]. In order to directly detect the bonding between NO and the iron atom and the reaction of NO upon photoactivation, resonance Raman spectra of the NHase were measured with 413 nm excitation at 85 K. Two prominent bands at 592 and 570 cm-1 were observed in the inactive from, and both of them were completely lost upon photoactivation. Upon subsequent introduction of 15NO, the active NHase was converted to the inactive form again and the above two bands were restored with downshifts by 10 and 12 cm-1, respectively. Also, the excitation profiles of these bands in the 350-500 nm region mostly followed the absorption spectrum arising from the iron center. From these isotopic shifts and the excitation profiles, the two Raman bands were assigned to the Fe-NO stretching and bending vibrations that are probably coupled with each other. The results provided solid evidence that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.

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Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6 M urea, and two kinds of subunits (alpha and beta) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400 nm and at 710 nm, which were characteristic of the activated enzyme. The beta subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before and after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4 degrees C for 1 h. Light irradiation of the beta subunit did not affect subsequent complex formation or NHase activity. However, the irradiated alpha subunit could not assemble with the beta subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the alpha subunit, and imply that light irradiation induces conformational change of the alpha subunit.

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The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m2/s at 310-400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.

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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.

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Cholesterol-5alpha,6alpha-epoxide hydrase activity was 96 percent greater in skin of hairless mice that were receiving suberythemic ultraviolet light irradiation for 15 weeks than in nonirradiated controls. This enzyme system, which metabolizes cholesterol-5alpha,6alpha-epoxide (a known carcinogen), appears to be substrateinducible and is apparently responsible for the concomitant reduction of the sterol carcinogen that occurs prior to tumor induction.

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