A gene cluster for chlorate metabolism in Ideonella dechloratans

Chlorate reductase has been isolated from the chlorate-respiring bacterium Ideonella dechloratans, and the genes encoding the enzyme have been sequenced. The enzyme is composed of three different subunits and contains molybdopterin, iron, probably in iron-sulfur clusters, and heme b. The genes (clr) encoding chlorate reductase are arranged as clrABDC, where clrA, clrB, and clrC encode the subunits and clrD encodes a specific chaperone. Judging from the subunit composition, cofactor content, and sequence comparisons, chlorate reductase belongs to class II of the dimethyl sulfoxide reductase family. The clr genes are preceded by a novel insertion sequence (transposase gene surrounded by inverted repeats), denoted ISIde1. Further upstream, we find the previously characterized gene for chlorite dismutase (cld), oriented in the opposite direction. Chlorate metabolism in I. dechloratans starts with the reduction of chlorate, which is followed by the decomposition of the resulting chlorite to chloride and molecular oxygen. The present work reveals that the genes encoding the enzymes catalyzing both these reactions are in close proximity.     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.     

The C subunit of Ideonella dechloratans chlorate reductase: expression, purification, refolding, and heme reconstitution

The C subunit of Ideonella dechloratans chlorate reductase has been expressed in Escherichia coli as a GST fusion protein. Purification from inclusion bodies, followed by refolding and reconstitution with heme, produced a protein with a heme/protein ratio of 0.4, and with UV-vis spectral characteristics similar to those of native chlorate reductase. Wavelength maxima for the alpha and beta bands in the reduced state were 559 and 529 nm for both native chlorate reductase and the reconstituted recombinant subunit, whereas the reduced Soret bands were found at 426 and 424 nm, respectively. These results support the notion of the C subunit as the cytochrome b moiety of I. dechloratans chlorate reductase. Moreover, the availability of a recombinant version of the C subunit is expected to facilitate further studies of electron transfer and protein interaction included in the reaction catalyzed by chlorate reductase.     

Comparison of native and recombinant chlorite dismutase from Ideonella dechloratans.

A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme. Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis. Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein. Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein. Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low pK(a). The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination. These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions.     

The primary structure of cytochrome c-554 from the green photosynthetic bacterium Chloroflexus aurantiacus.

The complete nucleotide sequence of the cytochrome c-554 gene from the green photosynthetic bacterium Chloroflexus aurantiacus has been determined. The derived amino acid sequence showed that the cytochrome precursor protein consists of 414 residues and contains 4-Cys-X-X-Cys-His- heme binding motifs. The only regions of the cytochrome c-554 sequence that were found to be significantly similar to the sequences of cytochromes from other organisms were the heme binding sites. The highest similarity was found with the heme binding segments in the four-heme reaction center cytochrome subunit from the purple photosynthetic bacterium Rhodopseudomonas viridis. The importance of this similarity for the evolutionary relationship between Chloroflexus and the purple bacteria is discussed.     

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min^–1 (mg protein)^–1. The dismutase obeyed Michaelis-Menten kinetics. The V _max and K _m calculated for chlorite were 2,200 U (mg protein)^–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30° C. Received: 9 April 1996 / Accepted: 12 August 1996     

Purification and characterization of spore-specific catalase-2 from Bacillus subtilis

Catalase-2, the catalase found in spores of Bacillus subtilis, has been purified to homogeneity from a nonsporulating strain. The apparent native molecular weight is 504,000. The enzyme appears to be composed of six identical protomers with a molecular weight of 81,000 each. The amino acid composition is similar to the composition of other catalases. Like most catalases, catalase-2 exhibits a broad pH optimum from pH 4 to pH 12 and is sensitive to cyanide, azide, thiol reagents, and amino triazole. The apparent Km for H2O2 is 78 mM. The enzyme exhibits extreme stability, losing activity only slowly at 93 degrees C and remaining active in 1% SDS-7 M urea. The green-colored enzyme exhibits a spectrum like heme d with a Soret absorption at 403 nm and a molar absorptivity consistent with one heme per subunit. The heme cannot be extracted with acetone-HCl or ether, suggesting that it is covalently bound to the protein.     

Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis

The beta-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH(2)-terminal amino acid sequence of the purified enzyme indicate that the beta-galactosidase subunit is composed of 1,038 amino acids with a calculated M(r) of 118,068. This beta-galactosidase shares structural properties with Escherichia coli beta-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis beta-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant beta-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis beta-galactosidase can outperform the current commercial beta-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted beta-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Nitric oxide-reductase homologue that contains a copper atom and has cytochrome c-oxidase activity from an aerobic phototrophic bacterium Roseobacter denitrificans

A cytochrome cb-type enzyme with cytochrome c-oxidase activity was purified from an aerobic phototrophic bacterium Roseobacter denitrificans. The enzyme was solubilized with sucrose monodecanoate from the membranes of R. denitrificans grown aerobically under light conditions, and purified to electrophoretic homogeneity. Absorption spectra of the purified enzyme showed peaks at 410 nm and 530 nm in the oxidized state, and peaks at 420, 522, and 551 nm and a shoulder at around 560 nm in the reduced state. The enzyme is composed of two subunits with apparent molecular weights on SDS-PAGE of 37,000 and 18,000, the latter positive to heme staining. The protein contains heme c, heme b, and copper in a 1:2:1 stoichiometry. The spectral properties indicated that the heme c and one heme b are in low-spin states, while the other heme b is in a high-spin state. The base sequences of the genes and the deduced amino acid sequences are similar to those of known NorB and NorC subunits of nitric oxide reductases from other bacterial species. The enzyme is similar to nitric oxide reductase, but differs in that it contains copper. Virtually no nitric oxide reductase activity was detected in the purified enzyme.     

Cu,Zn superoxide dismutase from chicken erythrocytes.

1. Copper, zinc superoxide dismutase (Cu,Zn SOD) has been purified to homogeneity from chicken erythrocytes by anion-exchange, immobilized metal affinity and size exclusion chromatography. 2. Molecular properties (amino acid composition, molecular mass, subunit composition and spec. act.) of the chicken enzyme are similar to those of a bovine erythrocyte Cu,Zn SOD. 3. The chicken and bovine enzymes are immunologically similar since antisera raised against each enzyme are cross-reactive.     

Role of the tetrahemic subunit in Desulfovibrio vulgaris hildenborough formate dehydrogenase

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH), the genome sequencing revealed the presence of three operons encoding formate dehydrogenases. fdh1 encodes an alphabetagamma trimeric enzyme containing 11 heme binding sites; fdh2 corresponds to an alphabetagamma trimeric enzyme with a tetrahemic subunit; fdh3 encodes an alphabeta dimeric enzyme. In the present work, spectroscopic measurements demonstrated that the reduction of cytochrome c(553) was obtained in the presence of the trimeric FDH2 and not with the dimeric FDH3, suggesting that the tetrahemic subunit (FDH2C) is essential for the interaction with this physiological electron transfer partner. To further study the role of the tetrahemic subunit, the fdh2C gene was cloned and expressed in Desulfovibrio desulfuricans G201. The recombinant FDH2C was purified and characterized by optical and NMR spectroscopies. The heme redox potentials measured by electrochemistry were found to be identical in the whole enzyme and in the recombinant subunit, indicating a correct folding of the recombinant protein. The mapping of the interacting site by 2D heteronuclear NMR demonstrated a similar interaction of cytochrome c(553) with the native enzyme and the recombinant subunit. The presence of hemes c in the gamma subunit of formate dehydrogenases is specific of these anaerobic sulfate-reducing bacteria and replaces heme b subunit generally found in the enzymes involved in anaerobic metabolisms.     

A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.     

Unique presence of a manganese catalase in a hyperthermophilic archaeon,Pyrobaculum calidifontis VA1

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.     

Purification and characterization of catalase from a facultative alkalophilic Bacillus

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).     

Characterization of a protein kinase from chicken oviductal fluids

Protein kinase has been found extracellularly in avian oviductal secretions. The enzyme has been isolated and shown to be primarily type II cAMP enhanced. The Ka for cAMP activation, binding and elution from ion-exchange columns, molecular weight of subunits, pH optimum as a histone kinase, response to cations, kinetic properties, and preference for lysine-rich histone are similar to those of mammalian type II protein kinase. Gel filtration data suggest that the dimer form is the functional entity in the reproductive tract. The catalytic subunit has been purified to greater than 90% homogeneity and has a Km of 4 mumol/l and Vmax of 10(6) U/mg, comparable to published values for bovine catalytic subunit.