Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.     

Purification and characterization of EDTA monooxygenase from the EDTA-degrading bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35 degreesC. Kms were 34.1 microM for uncomplexed EDTA and 8.5 microM for MgEDTA2-; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2-. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Cytochrome b5-like hemoprotein/cytochrome b5 reductase complex in rat liver mitochondria has NADH-linked aquacobalamin reductase activity

Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.     

Heterologous expression of carbonyl reductase: demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese superoxide dismutase.     

Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c-type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N-oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.     

The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action

Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.     

4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by N-ethylmaleimide-reductase of Yarrowia lipolytica

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.     

Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis

Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.     

Pyrroline-5-carboxylate reductase in human erythrocytes

Pyrroline-5-carboxylate reductase, which converts pyrroline-5-carboxylate to proline, has been identified in human erythrocytes. The level of pyrroline-5-carboxylate reductase activity in these cells is comparable to the activity levels of major erythrocyte enzymes. The physiologic function of the enzyme in erythrocytes cannot be related to its function in other tissues, i.e., producing proline for protein synthesis. We examined the kinetic properties of erythrocyte pyrroline-5-carboxylate reductase and compared them to the properties of the enzyme from proliferating cultured human fibroblasts. We found that the kinetic properties and regulation of the erythrocyte enzyme are distinctly different from those for human fibroblast pyrroline-5-carboxylate reductase. These characteristics are consistent with the interpretation that the function of the enzyme in human erythrocytes may be to generate oxidizing potential in the form of NADP+.     

Differential redox and electron-transfer properties of purified yeast, plant and human NADPH-cytochrome P-450 reductases highly modulate cytochrome P-450 activities

Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activities with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.     

Dental disease and current treatment needs in a group of physically handicapped children

The enzyme glutathione reductase (GR) (GSSG+NADPH+H+-->2 GSH+NADP+) plays a key role in the cellular defense against oxidative stress. High levels of GR activity are often associated with tumor growth and/or resistance mechanisms against drug and radiation therapy. In order to investigate the molecular basis of elevated glutathione reductase activities we studied the enzyme at the DNA, mRNA and protein levels in murine experimental tumor cell lines and in human lung tumors. A modified ultracentrifugation procedure was developed which allowed the simultaneous isolation of DNA and total cellular RNA. Out of 11 human bronchial carcinomas obtained from patients without prior chemotherapy, five tumors showed a GR activity which was 2.4 to 3.8 times higher than in the respective control tissues. In each case the elevated enzyme activity was accompanied by an elevated GRmRNA levels. For none of the tumors, GR gene rearrangement or amplification was observed by Southern blot analyses. The mouse tumor cell lines ASB XIV, Lewis lung carcinoma and EAT cells, also showed high levels of GRmRNA whereas this mRNA was hardly detectable in normal mouse lung tissue.     

Effect of Intermetallic Compounds on Microstructure and Mechanical Properties of Hot Roll Bonding Titanium to Steel

Titanium cladding on steel leads to the creation of properties such as resistance to corrosion that contribute to a widespread application of this metal composite in industries such as nuclear, chemical, aerospace, and biomaterial. One of the solid-state bonding methods to apply such a clad is to use the roll bonding method. In this paper, quality of the titanium cladded on carbon steel was studied in terms of metallurgical, mechanical properties and the effect of the copper interlayer on the metallurgical properties of bonding. The interface between the clad and the base metal was studied using a scanning electron microscope and light microscope and the phases formed were identified by X-ray diffraction analysis. The results showed that an increase in the bonding temperature increased the thickness of the intermetallic compounds, increased the hardness at close distances to the interface, and reduced the adhesion of the titanium cladded to the base metal.     

Sequence-alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B-276

Whole cells of Nocardia corallina B-276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH-dependent reductase (reductase) and a regulatory component (coupling protein). The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence-alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu-Xaa-Xaa-His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active-site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.     

Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.     

Acinetobacter radioresistens metabolizing aromatic compounds. 2. Biochemical and microbiological characterization of the strain

The metabolic potentialities of an Acinetobacter radioresistens strain, isolated from the soil adjacent to an activated sludge plant, were investigated. Among 26 aromatic substrates tested, only phenol, benzoate and catechol were metabolized. Since this strain possessed abundant plasmid DNA, the antibiotic and heavy metal resistance was examined, and the bacterial cells proved to be sensitive to all metals (Ni, Tl, Pb, Cd, Ag, Co, Zn) and antibiotics tested except for Fosfomycin and chloramphenicol. The degradation kinetics for phenol and benzoate as the sole carbon/energy source (pH 7, 30 degrees C) displayed different trends, confirmed by the bacterial growth curve. Crude extracts from phenol-grown cultures showed both phenol hydroxylating activity and catechol dioxygenating activity. Phenol hydroxylase possessed a reductase component able to reduce nitroblue tetrazolium (NBT) and cytochrome C, thus exhibiting differences from previously reported monocomponent phenol hydroxylases from the same genus. Catechol dioxygenase is an intradiol-cleaving enzyme recognizing also substituted catechols.     

Enzymology of mitomycin C metabolic activation in tumour tissue. Characterization of a novel mitochondrial reductase

In this study, the enzymology of mitomycin C (MMC) bioactivation in two murine colon adenocarcinomas, MAC 16 and MAC 26, was examined. Subcellular quinone reductase assessment via cytochrome c reduction confirmed a number of active enzymes. MAC 16 exhibited 22-fold greater levels of cytosolic DT-diaphorase than MAC 26, while microsomal NADPH:cytochrome P-450 reductase levels were similar in both tumour types. Metabolism of MMC by subcellular fractions isolated from both MAC 16 and MAC 26 was quantitated by monitoring the formation of the principle metabolite 2,7-diaminomitosene (2,7-DM) via high-performance liquid chromatography (HPLC). In MAC 16 only, activity displaying the properties of cytosolic DT-diaphorase and microsomal NADPH:cytochrome P-450 reductase was detected and confirmed, using the enzyme inhibitors dicoumarol and cytochrome P-450 reductase antiserum, respectively. The highest level of MMC metabolism was associated with the mitochondrial fraction from both tumours and was the sole enzyme activity detected in MAC 26. The greatest mitochondrial drug metabolism was achieved in the presence of NADPH as cofactor and hypoxia (MAC 16-specific activity, 3.67 +/- 0.58 nmol/30 min/mg; MAC 26 specific-activity, 3.87 +/- 0.71 nmol/30 min/mg) and was unaffected by the addition of the inhibitors dicoumarol and cytochrome P-450 reductase antiserum. NADH-dependent mitochondrial activity was only observed in MAC 16 at approximately 4-fold less than that seen with NADPH. MAC 26 homogenate incubations displayed enhanced metabolism under hypoxia, presumably due to the presence of the identified mitochondrial enzyme. MAC 16 homogenates showed no increase in metabolism under hypoxia, suggesting that other enzyme(s) may be predominant. These data indicate the presence of a novel mitochondrial one-electron reductase capable of metabolising MMC in MAC 16 and MAC 26.     

Guanosine monophosphate reductase from Artemia salina: Inhibition by xanthosine monophosphate and activation by diguanosine tetraphosphate

In the course of studies on the metabolic role of diguanosine tetraphosphate during development of Artemia salina, a guanosine monophosphate (GMP) reductase has been found in partially purified from the 150 000g Artemia cysts supernatant. From Lineweaver-Burk plots, two apparent Km values of 5 and 50 muM were obtained for GMP. Xanthosine monophosphate (XMP) is a very strong inhibitor of the reaction. In the presence of 1.5 muM XMP hyperbolic kinetics are found. Diguanosine tetraphosphate counteracts very effectively the inhibition of the activity by XMP, concomitantly changing to hyperbolic the kinetics of the enzyme, with a unique Km value of about 5 muM. The complex kinetic and the existence of allosteric e-fectors at physiological concentrations, together with our lack of success in resolving two isoenzymes, makes it very likely that GMP reductase presents negative cooperativity towards its substrate. The effect of diguanosine tetraphosphate on the enzyme is very specific; other structural analogues, diadenosine tetraphosphate and diguanosine triphosphate, tested a micromolar concentrations had no detectable effect on the enzyme. Guanosine triphosphate (GTP) (mM) was also able to counteract the inhibition of guanosine monophosphate (GMP) reductase by XMP. The properties of the Artemia GMP reductase are here compared with those of the similar enzyme from calf thymus and Escherchia coli. As a consequence, the regulation of eukaryotic GMP reductase is resulting to be quite different from that of the reductase from prokaryotes.     

The solution structure of parsley [2Fe-2S]ferredoxin

The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.     

In situ formation of multi scale reinforcing phases to improve performance of Al2O3-SiC C castables

In order to improve the physical properties and slag corrosion resistance of refractory materials for hot metal pretreatment, different contents of composite metal powders (CMP) were introduced into Al2O3-SiC-C refractory castables. The effect of CMP on the microstructure, physical properties and slag corrosion resistance of Al2O3-SiC-C refractory castables were studied. The results show that the multi scale reinforcing phases including flake crystals, rod shaped fibers, filamentous fibers and whiskers are in situ formed in the samples with addition of CMP, which result to the improvement of the cold modulus of rupture and cold crushing strength. When the addition of CMP is 6 wt.%, the high temperature modulus of rupture increases by 231%, the thermal shock resistance increases by 77% after thermal shock by water cooling 5 times and the percentage of the slag resistant area reduces by 37.2%.