Identification of the omega-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein requiring phospholipid for catalytic activity

Fatty acid and alkane hydroxylation by the inducible, cyanide-sensitive enzyme system of Pseudomonas oleovorans involves three enzymes: NADH-rubredoxin reductase, rubredoxin, and an ω-hydroxylase. The ω-hydroxylase is a yellow protein containing one atom of iron per polypeptide chain of molecular weight 42,000. Catalytic activity is lost upon removal of the iron and is restored when ferrous ions are incubated with the apohydroxylase. Hydroxylase preparations from which most of the phospholipid has been removed exhibit decreased activity unless supplemented with a phospholipid fraction from this bacterium or with dilauroylglyceryl-3-phosphorylcholine.     

Malate metabolism by Desulfovibrio gigas and its link to sulfate and fumarate reduction: purification of the malic enzyme and detection of NAD(P)+ transhydrogenase activity

Malate metabolism was investigated in lactate grown cells of Desulfovibrio gigas ; 3 mol of malate are converted into 2 mol succinate and 1 mol acetate. The malic enzyme (L-malate:NADP+ oxidoreductase) was purified to homogeneity and partially characterized. The enzyme is monomeric with molecular weight of 45 kDa. Its spectrum has no visible absorption and the activity is stimulated by K+ and Mg2+. The presence of an NAD(P)+ transhydrogenase, the observation of partial reduction of adenylylsulfate reductase by NADH (via NADH-rubredoxin oxidoreductase) and evidence for NADH-linked fumarate reductase activity support the involvement of pyridine nucleotides in the electron pathway toward the reduction of sulfur compounds and/or fumarate. An electron transfer chain to fumarate is proposed, taking into consideration these results and the stoichiometry of end-products derived from malate dismutation.     

Isolation and properties of reduced nicotinamide adenine dinucleotiderubredoxin oxidoreductase of Clostridium acetobutylicum

NADH-rubredoxin oxidoreductase from $\text{Clostridium acetobutylicum}$ was purified to yield a single band on disc-gel electrophoresis. The molecular weight was found to be 41 000 and did not dissociate into subunits when treated with sodium dodecylsulfate and 2-mercaptoethanol. The prosthetic group of the enzyme was identified as FAD. The enzyme reduces rubredoxine, ferricyanide dichlorophenolindophenol, p-toluoquinone, p-benzoquinone, 1,4-naphtoquinone but not ferredoxin or flavodoxin. The reaction does not occur when NADPH is substituted for NADH. The NADH-rubredoxin reductase was extremely sensitive to the thiol inhibitors.     

Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation.

Summary It had previously been held that chlorate is not itself toxic, but is rendered toxic as a result of nitrate reductase-catalysed conversion to chlorite. This however cannot be the explanation of chlorate toxicity in Aspergillus nidulans, even though nitrate reductase is known to have chlorate reductase activity. Among other evidence against the classical theory for the mechanism of chlorate toxicity, is the finding that not all mutants lacking nitrate reductase are clorate resistant. Both chlorate-sensitive and resistant mutants lacking nitrate reductase, also lack chlorate reductase. Data is presented which implicates not only nitrate reductase but also the product of the nirA gene, a positive regulator gene for nitrate assimilation, in the mediation of chlorate toxicity. Alternative mechanisms for chlorate toxicity are considered. It is unlikely that chlorate toxicity results from the involvement of nitrate reductase and the nirA gene product in the regulation either of nitrite reductase, or of the pentose phosphate pathway. Although low pH has an effect similar to chlorate, chlorate is not likely to be toxic because it lowers the pH; low pH and chlorate may instead have similar effects. A possible explanation for chlorate toxicity is that it mimics nitrate in mediating, via nitrate reductase and the nirA gene product, a shut-down of nitrogen catabolism. As chlorate cannot act as a nitrogen source, nitrogen starvation ensures.     

Purification and Characterization of Cytochrome P450 Reductase from Liver Microsomes of Feral Leaping Mullet (Liza saliens)

NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 μmol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 ± 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent K_m of the purified enzyme was found to be 7.69 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 μM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 103–113, 1998     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Inhibition of electron transfer in the cytochromeb-c 1 segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochromec reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochromec reductase and ubiquinol-cytochromec reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochromec reductase activities is markedly dependent on pH in the range pH 7–8. At pH 7.0 inhibition occurs with an apparentK _i1×10^–8 M, while at pH 8.0 the apparentK _i is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pK _a at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in theb-c ₁ segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a functional group in theb-c ₁ segment.     

Properties of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase in Villous and Crypt Cells of the Rat Small Intestine

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in microsomes of villous and crypt cells from the jejunal and ileal epithelia of rats fed commercial pellet were studied. The optimum pH of the microsomal reductase from villi and crypts was 7.0~7.2 and the Km for HMG-CoA was 41.7 µm. The reductase specifically required dithiothreitol for its activity. The activity was higher in ileal populations than in the jejunum. Responses of the reductase in the villous fraction to feeding cholesterol and taurocholate in combination or cholestyramine resembled those observed in crypt cells. Thus, the properties of microsomal HMG-CoA reductase in villous and crypt cells from the small intestine are similar each other, and they are possibly the same enzyme.     

Factors affecting in vivo nitrate reductase activity in triticale

Factors influencing in vivo nitrate reductase activity in triticale (×Triticosecale Wittmack) primary leaves were investigated. Nitrate reductase activity was found to be a function of reaction time or tissue weight. In the range of 1–10 mm, the optimum slice width for nitrate reductase activity in triticale was found to be 1–2 mm. The optimum exogenous nitrate concentration is 300 mM. Substantial nitrite production was obtained even when exogenous nitrate was omitted from the assay. Of the five low molecular weight organic solvents tested, n-propanol is the most effective in enhancing enzyme activity. The optimum n-propanol concentration is 1% (v/v). The concentration of phosphate buffer (pH 6) does not affect nitrate reductase activity. Enzyme activity drops significantly below or above pH 6. In our system, nitrite production is enhanced by incubating under nitrogen, instead of air. The highest level of in vivo activity of nitrate reductase was found to be 10–15 cm from tip, which is close to the basal meristem of triticale primary leaves. Younger but physiologically mature leaves have higher nitrate reductase activity than old leaves.     

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK _m andV _max of 76 M and 21 nmol·min^-1·(mg protein)^-1, respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.     

Impact of low pH and aluminium on nitrogen uptake and metabolism in roots of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

The effect of low pH and aluminum on nitrogen uptake and metabolism was studied in roots of Lotus japonicus grown in hydroponic cultures. The low pH slightly suppressed root elongation, and this effect was accompanied by the suppression of nitrate and ammonia uptake, as well as the nitrate reductase activity. In spite of high resistance of young Lotus plants to short-term Al application, the one-day treatment of Al strongly reduced nitrate uptake and also the activity of nitrate reductase (NRA) in the apical parts of roots. The glutamine synthetase activity was also suppressed by Al treatment, but in lower extent. On the other hand, the ammonium uptake and nitrite reductase activity stayed unchanged by Al treatment and the values were practically the same as in control plants. These results support the view that nitrate uptake and nitrate reduction might be the main processes responsible for Al induced growth retardation in Lotus plants grown in mineral acid soils.     

Localization,induction and characterization of a novel carbon–carbon double bond reductase from Aspergillus versicolor

Abstract

A novel reductase has been detected in cell-free extracts from growing/resting cultures of the fungus Aspergillus versicolor D-1, which specifically catalyzes NADPH-dependent reduction of the γ,δ-double bond of the lactone-conjugated unsaturated system in securinine to form 14,15-dihydrosecurinine. The localization of the reductase has been investigated using differential centrifugation techniques. It was found that the securinine reductase is a cytosolic enzyme. The reductase was highly inducible in growing/resting cultures when securinine was used as the substrate and inducer. Optimal incubation conditions for assay of the securinine reductase were determined by using the enzyme preparation from resting cultures of A. versicolor D-1. The optimum temperature and pH for the reductase activity were in the range of 20–24°C and 8.0–8.5 in 0.05 M Tris–HCl buffer, respectively. The thermal stability of the securinine reductase was poor.     

Soluble electron-transport activities in fresh and aged turnip tissue

Summary A soluble (supernatant) fraction from turnips catalyses the reduction of both FeCN and DCPIP but usually not cytochrome c in the presence of either NADH or NADPH. Slicing and aging turnip tissue induces an increase in these activities as well as the development of an NADH-cytochrome c reductase activity.(NH₄)₂SO₄ and Sephadex fractionation indicated that at least three enzymes were involved: an NADH-cytochrome-c reductase, an NADH-FeCN reductase, and an NAD(P)H-DCPIP and FeCN reductase. While the latter reductase had an acid pH optimum, indicating vacuolar origin, the NADH-cytochrome-c and FeCN reductases both had neutral pH optima, indicating cytoplasmic origin. Characterization of the NADH-specific reductases indicated that NADH-FeCN reductase may be a soluble form of the microsomal membrane NADH dehydrogenase but that NADH-cytochrome-c reductase may be normally soluble and possibly involved in cyanide-sensitive NADH oxidation.The induced development of all three reductases was inhibited by 6-methylpurine, ethionine and cycloheximide, indicating dependence on both RNA and protein synthesis. The inhibition by cycloheximide could be reversed but this reversion required a 20-h washing-out period to be complete.Abbreviations DCPIP 2,6-dichlorophenol indophenol - FeCN ferricyanide - NO QNO 2-n-nonylhydroxyquinoline-N-oxide - pCMB p-chloromercuribenzoate - SF soluble fraction     

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed.     

Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants

Summary Mutants of A. nidulans at several loci lack detectable NADPH-nitrate reductase activity. These loci include niaD, the structural gene for the nitrate reductase polypeptide, and five other loci termed cnxABC, E, F, G and H which are presumed to be involved in the formation of a molybdenum-containing component (MCC) necessary for nitrate reductase activity. When frozen mycelia from A. nidulans deletion mutant niaD26 were homogenized in a Ten Broeck homogenizer together with frozen mycelia from either enzA6, cnxE29, cnxF12, enxG4 or cnxH3 strains grown on urea+nitrate as the nitrogen source, nitrate reductase activity was detectable in the extract. Similar results were obtained by co-homogenizing niaD mycelia with Neurospora crassa nit-1 mycelia induced on nitrate. Thus, all A. nidulans cnx mutants are similar to the N. crassa nit-1 strain in their capacity to yield NADPH-nitrate reductase in the presence of the presumed MCC. As judged by the amounts of nitrate reductase formed, niaD26 mycelia grown on urea±nitrate contained much more available MCC than ammonium-grown mycelia. No NADPH-nitrate reductase activity was found in extracts prepared by co-homogenizing mycelia from all five A. nidulans cnx strains. Wild-type A. nidulans NADPH-nitrate reductase acid dissociated by adjustment to pH 2.0–2.5 and re-adjusted to pH 7 could itself re-assemble to form active nitrate reductase and thus was not a sueful source of MCC for these experiments. These results are consistent with the conclusion that the active nitrate reductase complex is composed of polypeptide components which are the niaD gene product, plus the MCC which is formed through the combined action of the cnx gene products. Further, the production of MCC may be regulated in response to the nitrogen nutrition available to the organism.     

Effects of nitrate,ammonium and pH on the growth of conifer seedlings and their production of nitrate reductase

Summary Lodgepole pine (Pinus contorta Dougl.), Engelmann spruce (Picea engelmanni Parry), and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown in open-ended tube cultures of sand and perlite, irrigated with nitrate, ammonium, and a 1∶1 mixture of ammonium and nitrate, combined factorially with pH values of 4.6, 5.3 and 6.0 giving a total of nine treatments. Douglas-fir showed intolerance to ammonium which was especially marked in root weight. Lodgepole pine and Engelmann spruce made poor growth with nitrate, but showed little difference between ammonium and mixed sources. Only Douglas-fir showed a significant response to pH treatments with pH 5.3 plants being largest. Contamination of the sand with carbonate-bicarbonate, apparently caused seedlings grown in ammonium solutions to be larger in sand than in perlite. Douglas-fir grown in perlite cultures showed a growth response like the first experiment and nitrate reductase activity in the order nitrate > nitrateammonium mixture > ammonium. Plastic bead cultures had poor growth response due to low retention of water by the substrate, but the nitrate reductase assays produced results like the perlite cultures. Lodgepole pine grown in water culture demonstrated the well known pH shift associated with different nitrogen forms, and when assayed for nitrate reductase these seedlings had larger relative activities than Douglas-fir, but the order of activity remained nitrate > mixed source > ammonium.     

In vivo Characterization of Nitrate Reductase Activity in Cotton

The in vivo nitrate reductase activity in leaf tissue of cotton (Gossypium hirsutum L.) was characterized. Enzymatic activity was linear with time up to 60 min. The assay for nitrate reductase activity was optimized in leaf slices 400 μm wide incubated in an anaerobic system at 30°C, in a 0.02 M KNO₃ medium at pH 7.0 with 1 % propanol. In vivo activity was highest in recently matured leaves at the top of the plant. Both light and nitrate enhanced in vivo enzymatic activity. The activity was highest after 9 hours in the light and then decreased steadily for several more hours even in the presence of light. The nitrate reductase activity was more strongly correlated to the levels of NO₃-N in the culture solution than to the NO₃-N level in the tissue. The utility of this technique in nitrate reductase assay in a tissue containing large amounts of phenolic compounds is discussed.     

Expression and Characterization of the HMG-CoA Reductase of the Thermophilic Archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA^AGA,AGG, the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA^AGA,AGG resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K_i for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.     

Effects of Exogenous Proline and Glycinebetaine on the Salt Tolerance of Rice Cultivars

Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100,000 and was composed of two identical subunits of molecular weight (M_rs, 52,000) as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125 μM, respectively.