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1.
Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase. 总被引:15,自引:0,他引:15
Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil. 相似文献
2.
Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.
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Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. 相似文献
3.
Miseviciene L Anusevicius Z Sarlauskas J Harris RJ Scrutton NS Cenas N 《Acta biochimica Polonica》2007,54(2):379-385
In order to clarify the poorly understood mechanisms of two-electron reduction of quinones by flavoenzymes, we examined the quinone reductase reactions of a member of a structurally distinct old yellow enzyme family, Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase (PETNR). PETNR catalyzes two-electron reduction of quinones according to a 'ping-pong' scheme. A multiparameter analysis shows that the reactivity of quinones increases with an increase in their single-electron reduction potential and pK(a) of their semiquinones (a three-step (e(-),H(+),e(-)) hydride transfer scheme), or with an increase in their hydride-transfer potential (E(7)(H(-))) (a single-step (H(-)) hydride transfer scheme), and decreases with a decrease in their van der Waals volume. However, the pH-dependence of PETNR reactivity is more consistent with a single-step hydride transfer. A comparison of X-ray data of PETNR, mammalian NAD(P)H : quinone oxidoreductase (NQO1), and Enterobacter cloacae nitroreductase, which reduce quinones in a two-electron way, and their reactivity revealed that PETNR is much less reactive, and much less sensitive to the quinone substrate steric effects than NQO1. This may be attributed to the lack of pi-pi stacking between quinone and the displaced aromatic amino acid in the active center, e.g., with Phe-178' in NQO1. 相似文献
4.
Khan H Barna T Harris RJ Bruce NC Barsukov I Munro AW Moody PC Scrutton NS 《The Journal of biological chemistry》2004,279(29):30563-30572
The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed. 相似文献
5.
A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently. 相似文献
6.
Basran J Harris RJ Sutcliffe MJ Scrutton NS 《The Journal of biological chemistry》2003,278(45):43973-43982
The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme. 相似文献
7.
8.
Kinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23,500 with a sedimentation coefficient of 6.0S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor. 相似文献
9.
Crystallographic analysis of the binding of NADPH, NADPH fragments, and NADPH analogues to glutathione reductase 总被引:6,自引:0,他引:6
The binding of the substrate NADPH as well as a number of fragments and derivatives of NADPH to glutathione reductase from human erythrocytes has been investigated by using X-ray crystallography. Crystals of the enzyme were soaked with the compounds of interest, and then the diffraction intensities were collected out to a resolution of 3 A. By use of phase information from the refined structure of the native enzyme in its oxidized state, electron density maps could be calculated. Difference Fourier electron density maps with coefficients Fsoak - Fnative showed that the ligands tested bound either at the functional NADPH binding site or not at all. Electron density maps with coefficients 2Fsoak - Fnative were used to estimate occupancies for various parts of the bound ligands. This revealed that all ligands except NADPH and NADH, which were fully bound, showed differential binding between the adenine end and the nicotinamide end of the molecule: The adenine end always bound with a higher occupancy than the nicotinamide end. Models were built for the protein-ligand complexes and subjected to restrained refinement at 3-A resolution. The mode of binding of NADPH, including the conformational changes of the protein, is described. NADH binding is clearly shown to involve a trapped inorganic phosphate at the position normally occupied by the 2'-phosphate of NADPH. A comparison of the binding of NADPH with the binding of the fragments and analogues provides a structural explanation for their relative binding affinities. In this respect, proper charge and hydrogen-bonding characteristics of buried parts of the ligand seem to be particularly important. 相似文献
10.
T M Barna H Khan N C Bruce I Barsukov N S Scrutton P C Moody 《Journal of molecular biology》2001,310(2):433-447
Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme. 相似文献
11.
C E Grimshaw M Shahbaz G Jahangiri C G Putney S R McKercher E J Mathur 《Biochemistry》1989,28(13):5343-5353
Aldose reductase, purified to homogeneity from bovine kidney, is converted in a temperature-dependent process from a low-Km/low-Vmax form to a high-Km/high-Vmax form of the enzyme. Activation, which results in significant changes in the protein secondary structure, as detected by fluorescence spectroscopy, circular dichroism, and thiol modification with 5,5'-dithiobis(2-nitrobenzoic acid), has no effect on the apparent Mr, pI, or homogeneity of the enzyme, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose isoelectric focusing. Vmax, which varied less than 3-fold for a series of aldehyde substrates with either activation state of the enzyme, increased an average of (17 +/- 4)-fold upon activation of the enzyme. V/Kaldehyde increased or decreased up to 4-fold, depending on the substrate. Activation desensitized the enzyme to inhibition by aldose reductase inhibitors, with the apparent Ki value increasing from 2-fold for Epalrestat [ONO-2235, (E)-3-(carboxymethyl)-(E)-5-[2-methyl-3-phenylpropenylidene]-rhoda nine] to 200-fold for AL-1576 (spiro [2,7-difluorofluorene-9,4'-imidazolidine]-2',5'-dione). Biphasic double-reciprocal plots for the aldehyde substrates and biphasic Dixon plots for inhibition by AL-1576 and Statil [ICI-128,436; 3-[(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-l-ylacetic acid], observed during the course of activation, are quantitatively accounted for by the individual contributions of the two enzyme forms. On the basis of an analysis of the kinetic data, a mechanism is proposed in which isomerization of the free enzyme limits the rate of the forward reaction for the unactivated enzyme and is the primary step affected by activation. 相似文献
12.
Kinetic and structural characterization of the glutathione-binding site of aldose reductase 总被引:2,自引:0,他引:2
Dixit BL Balendiran GK Watowich SJ Srivastava S Ramana KV Petrash JM Bhatnagar A Srivastava SK 《The Journal of biological chemistry》2000,275(28):21587-21595
Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase. 相似文献
13.
14.
The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme-progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitroaromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis. Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme. The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation. We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of alpha/beta unsaturated carbonyl compounds. The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer. With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180 degrees , consistent with known geometries for hydride transfer in flavoenzymes. Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies. 相似文献
15.
D K Apps 《European journal of biochemistry》1975,55(2):475-483
NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo. 相似文献
16.
NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid. 相似文献
17.
Rastelli G Sirawaraporn W Sompornpisut P Vilaivan T Kamchonwongpaisan S Quarrell R Lowe G Thebtaranonth Y Yuthavong Y 《Bioorganic & medicinal chemistry》2000,8(5):1117-1128
The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance. 相似文献
18.
Kinetic and structural studies of hydroxyproline 2-epimerase 总被引:2,自引:0,他引:2
19.
Çakmak R Durdagi S Ekinci D Sentürk M Topal G 《Bioorganic & medicinal chemistry letters》2011,21(18):5398-5402
Discovery of GR inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, the synthesis and GR inhibitory capacities of novel nitroaromatic compounds (NCs) (1-3) were reported. Some commercially available molecules were also tested for comparison reasons. The novel NCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low micromolar concentrations with K(i) values ranging from 0.211 to 4.57 μM as compared with well-known agents. Inhibition mechanism was assessed as being due to occlusion of the active site entrance by means of the NCs. Molecular docking results have shown that docking poses of ligands are able to construct binding interactions with the essential amino acids. 相似文献
20.
NADPH and NADP+ levels were measured in rat lens from normal controls, from galactose-fed and diabetic rats during the first week of cataract formation. The level of NADPH in normal rat lens was determined to be 12.3 +/- 0.4 nmol/g wet weight, and that of NADP+ 4.6 +/- 0.2 nmol/g wet weight. In early cataract formation NADPH levels decreased rapidly during the first 2 days and then remained stable at 76% of control for galactose-fed and 84% for diabetic rats. NADP+ levels increased by 38% of control for galactose-fed and 54% for diabetic rats. Calculated NADPH/NADP+ ratios dropped from 3.36 +/- 0.21 to 1.86 +/- 0.16 in galactose fed rats, and from 2.81 +/- 0.15 to 1.61 +/- 0.16 in diabetic rats (P less than 0.001 for both experimental groups). These data are consistent with rapid NADPH oxidation during onset of lens cataracts. No significant changes in aldose reductase enzymatic activity levels were observed in either the galactosemic or the diabetic rats during the times measured. 相似文献