Cloning, purification and characterization of novel Cu-containing nitrite reductase from the Bacillus firmus GY-49.

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K m and K cat of NirK on sodium nitrite were 0.27 mM and 0.36 × 103 s-1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.

[Peroxidase activity of octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus].

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s­1; k cat/K m = 855 mM­1 s­1) has been 100­300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

Why orange Guaymas Basin Beggiatoa spp. are orange: single-filament-genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities.

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of GK0767, the copper-containing nitrite reductase from Geobacillus kaustophilus.

The soluble region (residues 32-354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426, has been cloned and overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.3 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5 Å. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V(M) value of 3.14 Å(3) Da(-1).

Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.

Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at WO(4)(2-) concentrations that are inhibitory to bacterial Nars. In this report, we establish that the enzyme purified from cells grown with 4.5 µM WO(4)(2-) contains W as the metal cofactor but is otherwise identical to the Mo-Nar previously purified from P. aerophilum grown at low WO(4)(2-) concentrations. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor. The W-Nar has a 2-fold lower turnover number (633 s(-1)) but the same K(m) value for nitrate (56 µM) as the Mo-Nar. Quinol reduction and nitrate oxidation experiments monitored by EPR with both pure W-Nar and mixed W- and Mo-Nar preparations suggest a monodentate ligation by the conserved Asp241 for W(V), while Asp241 acts as a bidentate ligand for Mo(V). Redox titrations of the Mo-Nar revealed a midpoint potential of 88 mV for Mo(V/IV). The E(m) for W(V/IV) of the purified W-Nar was estimated to be -8 mV. This relatively small difference in midpoint potential is consistent with comparable enzyme activities of W- and Mo-Nars. Unlike bacterial Nars, the P. aerophilum Nar contains a unique membrane anchor, NarM, with a single heme of the o(P) type (E(m) = 126 mV). In contrast to bacterial Nars, the P. aerophilum Nar faces the cell's exterior and, hence, does not contribute to the proton motive force. Formate is used as a physiological electron donor. This is the first description of an active W-containing Nar demonstrating the unique ability of hyperthermophiles to adapt to their high-WO(4)(2-) environment.

A eukaryotic copper-containing nitrite reductase derived from a NirK homolog gene of Aspergillus oryzae.

We cloned a bacterial copper-containing nitrite reductase (NirK) homolog gene of Aspergillus oryzae (AonirK). Alignment showed that amino acid residues crucial for copper binding are conserved in the deduced sequence of the fungal protein. The recombinant protein exhibited distinct nitrite reductase activity, and its absorption and EPR spectra showed the presence of type 1 and type 2 copper atoms in the molecule. AonirK transcriptionally responded to denitrification conditions. Although the denitrifying activity of A. oryzae was weak under the conditions employed, high expression of the gene in the fungal cells enhanced the denitrifying activity 6-fold, accompanied by distinct cell growth. Furthermore, the highly expressed AoNirK was subcellularly localized to the mitochondria. The results demonstrated that AoNirK is responsible for fungal denitrification. Discussion is added on the novel insight concerning the origin and evolution of the mitochondrion provided by the findings for eukaryotic NirKs.

Directed evolution of copper nitrite reductase to a chromogenic reductant.

Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.

Intersite structural rearrangement of the blue copper site induced by substrate binding: spectroscopic studies of a copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015.

A copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015 has its own unique blue or type 1 copper protein resonance Raman spectrum in the usual Cu-S(Cys) stretching region, nu(Cu-S(Cys)), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-S(Cys)) Raman bands at 412, 420, and 364 cm(-1) of the type 1 copper site all shifted to higher frequencies upon binding of nitrite to the type 2 copper site, and the resonance Raman difference spectra progressively intensified with the increments of nitrite ion concentration. Positive support for substrate binding to the type 2 copper is provided by the nu(Cu-S(Cys)) bands in the resonance Raman spectrum of a type 2 copper-depleted enzyme, which is insensitive to the presence of NO2-. The shift to higher frequency of the Raman bands of the type 1 copper center with the addition of nitrite ions suggests a stronger Cu-S(Cys) interaction in the substrate-bound A. xylosoxidans nitrite reductase.

Purification, characterization, and gene cloning of thermophilic cytochrome cd1 nitrite reductase from Hydrogenobacter thermophilus TK-6.

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can grow autotrophically under anaerobic conditions by denitrification. One of the denitrification enzymes, cytochrome cd(1) nitrite reductase, was isolated and its gene was cloned from strain TK-6. The subunit molecular mass of the purified enzyme was 61.5 kDa and the isoelectric point was determined to be 9.3. The optimum temperature and pH for the enzymatic reaction were 70-75 degrees C and 6.5-7.0, respectively. The structural gene for the enzyme, nirS, is probably transcribed as a hexacistronic operon with the following genes encoding a putative diheme cytochrome c and the proteins required for biosynthesis of heme d(1). The NirS sequence was phylogenetically distinct from those of proteobacteria. The consensus -35 and -10 sequences were found in the putative nirS promoter region, but the consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide sensing regulators were not found in this region.

The effects of salinity and other factors on nitrite reduction by Ochrobactrum anthropi 49187.

The nitrite reductase (NIR) gene was cloned from Ochrobactrum anthropi 49187 and found to contain an open reading frame of 1131 nucleotides, encoding a polypeptide of 376 amino acids. The O. anthropi NIR gene encodes a copper-type dissimilatory reductase based on sequence homology with other genes. The polypeptide product is predicted to form a trimeric holoenzyme of 37 kDa subunits based on molecular weight estimates of extracts in activity gels. Expression of the enzyme is up-regulated by nitrate, presumably through the intermediate nitrite, and its activity is influenced by inhibitors. Salinity enhances the activity of existing NIR enzyme, but appears to decrease the expression of new enzyme.

Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein.

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.

Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa.

The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.

The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase.

Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water. Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions. The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions. This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme. In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal. The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200. After oxidation, the iron moves into the d(1) heme plane. A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite. The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.

Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei.

The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised. H. mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source. The overall purification was 46-fold with about 4% recovery of activity. The enzyme is a monomeric protein of approximately 66 kDa. A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity. Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin. NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H. mediterranei.

The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli.

Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre. The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69. Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here. The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1).

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding.

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.

Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774.

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens.

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K(m )values for nitrate and nitrite were determined to be 32 and 10 microM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (-207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry.