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1.
To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.  相似文献   

2.
The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-14C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of ~0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O2, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP+. Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The m/z 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the m/z 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.  相似文献   

3.
2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities but also after munitions firing/detonation or leakage from explosive remnants of war. Due to its toxicity and recalcitrance, life cycle of TNT-containing products and bioremediation are critical issues. As TNT is a strongly electron-deficient aromatic with a positive molecular quadrupole moment and three electrophilic nitro groups, its environmental fate is contingent upon specific sorptive electron donor–acceptor interactions and nucleophilic, reductive (bio)transformations. The microbial degradation of TNT is governed by cometabolism and therefore depends on the growth substrate(s) available in contaminated environments. Long considered an ecotoxicological safety endpoint, the immobilization of TNT metabolites derived from nitro moiety reduction in soil is controversial because they preferentially bind to the dissolved soil organic matter which can be released into surface and groundwaters. The ever-growing biochemical knowledge of TNT degradation has made bioaugmentation and phytoremediation attractive alternatives. While the discovery and engineering of microorganisms with novel/improved degradative abilities are very challenging, the deciphering of the physiological roles of promiscuous enzymes involved in TNT biodegradation, such as type II hydride transferases of the Old Yellow Enzyme family, opens new perspectives for bioremediation. Finally, transgenic plants have enabled effective phytoremediation at the field scale, which is emerging as the preferable in situ option to rehabilitate TNT-contaminated sites.  相似文献   

4.
Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform, 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0–8.0 leads to the mono-and dihydride complexes of TNT (H?-TNT and 2H?-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 μM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 μM). Together with accumulation of nitrites (up to 76.0 μM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4-and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.  相似文献   

5.
Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.  相似文献   

6.
The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) are toxic environmental pollutants. The biotransformation and detoxication of these persistent compounds in higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathione transferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformation of TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities with both substrates.  相似文献   

7.
Our recent study highlights the role of 2 glutathione transferases (GSTs) in the detoxification of the environmental pollutant, 2,4,6-trinitrotoluene (TNT) in Arabidopsis thaliana. TNT is toxic and highly resistant to biodegradation in the environment, raising both health and environmental concerns. Two GSTs, GST-U24 and GST-U25, are upregulated in response to TNT treatment, and expressed predominantly in the root tissues; the site of TNT location following uptake. Plants overexpressing GST-U24 and GST-U25 exhibited significantly enhanced ability to withstand and detoxify TNT, and remove TNT from contaminated soil. Analysis of the catalytic activities of these 2 enzymes revealed that they form 3 TNT-glutathionyl products. Of particular interest is 2-glutathionyl-4,6-dinitrotoluene as this represents a potentially favorable step toward subsequent degradation and mineralization of TNT. We demonstrate how GSTs fit into what is already known about pathways for TNT detoxification, and discuss the short and longer-term fate of TNT conjugates in planta.  相似文献   

8.
The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.Large amounts of land and water are heavily contaminated by explosives, mainly as a result of the manufacture and military use of munitions. The high financial cost associated with cleaning up these contaminated sites largely precludes the use of many existing remediation technologies, such as soil excavation and incineration or disposal to landfill. There is a great deal of work documenting the global contamination, general toxicity, and microbial metabolism of 2,4,6-trinitrotoluene (TNT) in the environment; however, relatively little is known about the enzymes mediating the detoxification of TNT in plants (for review, see Rylott and Bruce, 2009).Phytoremediation, the use of plants to remove environmental pollutants, offers a low-cost, sustainable alternative to conventional remediation technologies and is attracting considerable attention as a means to clean up sites contaminated with explosives. While TNT is a potent phytotoxin, plants are able to detoxify low levels of TNT. In an effort to determine how plant tolerance could be further improved, we are investigating the biochemistry and enzymology underlying the innate ability of plants to detoxify TNT. The detoxification of xenobiotics has been loosely categorized into three phases (Sandermann, 1992): activation, conjugation, and compartmentation. The proposed route of TNT detoxification follows these phases. The electron-withdrawing properties of the nitro groups of TNT make the aromatic ring electron deficient. This favors reductive transformation reactions in plants, and the TNT molecule is most commonly activated by the reduction of a nitro group to give hydroxylamino and then amino derivatives (Fig. 1, pathway A). Following the introduction of a functional group, more hydrophilic molecules such as Glc are conjugated to the activated TNT molecule (Gandia-Herrero et al., 2008), facilitating transport and subsequent compartmentation or sequestration.Open in a separate windowFigure 1.The transformation of TNT by pentaerythritol tetranitrate reductase. Pathway A shows the transformation of the nitro group to nitroso-dinitrotoluene (NODNT), HADNT, and then ADNT products. Pathway B shows the reduction of the aromatic ring to form hydride and dihydride Meisenheimer complexes, then chemical condensation with HADNT to form diarylamines.Data from both our microarray experiments (Gandia-Herrero et al., 2008) and other expression studies (Ekman et al., 2003; Mezzari et al., 2005) have found that members of the small gene family of oxophytodienoate reductases (OPRs) in Arabidopsis (Arabidopsis thaliana) are up-regulated following exposure to TNT. The Arabidopsis genome contains three characterized OPRs: OPR1, OPR2, and OPR3. In addition, there are three as yet uncharacterized putative OPRs, named here as OPR4, OPR5, and OPR6, with OPR4 and OPR5 being identical. The physiological functions of the OPRs remain obscure, with the exception of OPR3, which is involved in jasmonic acid biosynthesis, converting (9S,13S)-12-oxophytodienoic acid to 3-2(2′(Z)-pentyl)cyclopentane-1-octanoic acid in the peroxisome (Sanders et al., 2000; Stintzi and Browse, 2000; for review, see Wasternack, 2007). OPR3 is located on chromosome II within the Arabidopsis genome and contains a C-terminal Ser-Arg-Leu type 1 peroxisome-targeting sequence. The remaining OPRs are all located on chromosome I and do not possess any known organelle-targeting sequences. The spatial expression of OPR1 and OPR2 across root cells where TNT accumulates (Biesgen and Weiler, 1999; Baerenfaller et al., 2008) favors a role in detoxification.The OPRs share similarity with the Old Yellow Enzyme family, a group of flavoenzymes that has been repeatedly associated with the transformation of explosives (Binks et al., 1996; Schaller and Weiler, 1997; Snape et al., 1997; Basran et al., 1998; French et al., 1998; Blehert et al., 1999; Pak et al., 2000; Fitzpatrick et al., 2003; Williams et al., 2004). Studies also indicate that Old Yellow Enzyme homologs function as antioxidants, detoxifying the breakdown products of lipid peroxidation and other toxic electrophilic compounds (Kohli and Massey, 1998; Williams and Bruce, 2002; Fitzpatrick et al., 2003; Trotter et al., 2006). This oxidative stress could result from exposure to xenobiotics including TNT, wounding, or pathogen attack.Pentaerythritol tetranitrate reductase, an Old Yellow Enzyme homolog isolated from Enterobacter cloacae (Binks et al., 1996), possesses two catalytic activities toward TNT (Fig. 1): nitroreduction of TNT to form hydroxylamino-dinitrotoluene (HADNT) and then amino-dinitrotoluene (ADNT), and aromatic ring reduction of TNT to yield hydride and dihydride (2H-TNT) Meisenheimer TNT adducts (French et al., 1998; Williams et al., 2004). The TNT ring-reduced compounds condense via a nonenzymatic reaction with HADNTs to form diarylamines, with the liberation of nitrite (Wittich et al., 2008). Expression of pentaerythritol tetranitrate reductase in tobacco (Nicotiana tabacum) confers both resistance to, and the ability to transform, TNT (French et al., 1999). OPR1, OPR2, and OPR3 share 43%, 44%, and 36% identity, respectively, with pentaerythritol tetranitrate reductase, and all possess the conserved active site amino acids crucial for TNT transformation by pentaerythritol tetranitrate reductase and other members of the Old Yellow Enzyme family (Snape et al., 1997; French et al., 1998; Khan et al., 2004), suggesting that they are capable of transforming TNT.The OPR4/5 protein is predicted to have reduced activity toward TNT, compared with the other OPRs, owing to a C-terminal truncation that removes residues thought to be important in binding the cofactor NADH, Thus, we investigated OPR1, -2, and -3 as likely candidates for the TNT nitroreduction activity in Arabidopsis.  相似文献   

9.
Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.  相似文献   

10.
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.  相似文献   

11.
The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R2 = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.  相似文献   

12.
《Phytochemistry》1987,26(6):1607-1611
Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[3H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.  相似文献   

13.
Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.  相似文献   

14.
Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 310 helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.  相似文献   

15.
It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.  相似文献   

16.
Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent Km and kcat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s−1, respectively. Cyanide, an inhibitor for the CO/CO2 oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.  相似文献   

17.
《Phytomedicine》2015,22(9):829-836
BackgroundBalanites aegyptiaca Del. (Zygophyllaceae) fruits are used to treat hyperglycemia in Egyptian folk medicine and are sold by herbalists in the Egyptian open market for this purpose. Nevertheless, the fruits have not yet been incorporated into pharmaceutical dosage forms. The identity of the bioactive compounds and their possible mechanisms of action were not well understood until now.PurposeAldose reductase inhibitors are considered vital therapeutic and preventive agents to address complications caused by hyperglycemia. The present study was carried out to identify the primary compounds responsible for the aldose reductase inhibitory activity of Balanites aegyptiaca fruits.Study designThe 70% ethanolic extract of Balanites aegyptiaca fruit mesocarp and its fractions were screened for inhibition of the aldose reductase enzyme. Bio-guided fractionation of the active butanol fraction was performed and the primary compounds present in the saponin-rich fraction (D), which were responsible for the inhibitory activity, were characterized. HPLC chromatographic profiles were established for the different fractions, using the isolated compounds as biomarkers.MethodsAldose reductase inhibition was tested in vitro on rat liver homogenate. The butanol fraction of the 70% ethanolic extract was fractionated using vacuum liquid chromatography (VLC, RP-18 column). The most active sub-fraction D, which was eluted with 75% methanol, was subjected to preparative HPLC to isolate the bioactive compounds.ResultsThe butanol fraction displayed inhibitory activity against the aldose reductase enzyme (IC50 = 55.0 ± 6 µg/ml). Sub-fraction D exhibited the highest inhibitory activity (IC50 = 12.8 ± 1 µg/ml). Five new steroidal saponin derivatives were isolated from this fraction. The isolated compounds were identified as compound 1a/b, a 7:3 mixture of the 25R:25S epimers of 26-O-β-D-glucopyranosyl-furost-5-ene-3,22,26-triol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 2, 26-O-β-D-glucopyranosyl-(25R)-furost-5-ene-3,22,26-triol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 3, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 4, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; and compound 5, which is the 25S epimer of compound 4, by using various spectroscopic methods [MS,1D and 2D NMR (HSQC, HMBC, DQF-COSY, HSQC-TOCSY)]. Compounds 1a/b, 2, 3, 4, 5 exhibited highly significant aldose reductase inhibitory activities (IC50 values were 1.9 ± 0.2, 1.3 ± 0.5, 5.6 ± 0.2, 5.1 ± 0.4, 5.1 ± 0.6 µM, respectively) as compared to the activity of the reference standard quercetin (IC50 = 6.6 ± 0.3 µM).ConclusionThe aldose reductase inhibitory activity of Balanites fruits is due to the steroidal saponins present. HPLC chromatographic profiles of the crude butanol fraction and its 4 sub-fractions showed that the most highly bioactive fraction D contained the highest amount of steroidal saponins (75%) as compared to the 21% present in the original butanol fraction. The isolated furostanol saponins proved to be highly active in an in vitro assay.  相似文献   

18.
The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate, and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC 824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high-solvent-producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only ~30 % of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70 % that of the parent. Overexpression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution, and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid-producing culture decreased from ?370 to ?200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 min. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate. The results show that during TNT transformation, the cells shift metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors generate a higher proportion of butyrate.  相似文献   

19.
Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.

Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.

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20.
The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Δ32-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10–25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through β-oxidation was more severely reduced in mutants devoid of Δ32-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Δ32-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.  相似文献   

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