植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生, 其中包括超氧化物歧化酶(SOD, EC1.15.1.1)、抗坏血酸过氧化物酶(APX, EC1.11.1.11)、过氧化氢酶(CAT, E.C.1.11.1.6 )和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9)。它们在清除活性氧过程中起着不同的作用。GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少。最近几年研究表明, 植物体内也存在类似于哺乳动物的GPXs家族, 并对其功能研究已初见端倪。本文综述了有关GPXs的结构以及植物GPXs功能的研究进展。     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

Selenium-independent epididymis-restricted glutathione peroxidase 5 protein (GPX5) can back up failing Se-dependent GPXs in mice subjected to selenium deficiency

We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs.     

Extracellular glutathione peroxidase from the blood‐sucking bug,Rhodnius prolixus

Glutathione peroxidase (GPX) activity was measured in several tissues of the blood‐sucking bug, Rhodnius prolixus. In contrast to the pattern found in vertebrates, where GPX is predominantly intracellular, the highest levels of this enzyme in Rhodnius were found in the hemolymph. The hemolymph glutathione‐dependent peroxidase accepted both H₂O₂ and t‐butyl hydroperoxide as substrates. This fact, together with the absolute glutathione dependence, inhibition by mercaptosuccinate, insensitivity to cyanide, and a molecular mass (100.7 kDa) similar to vertebrate GPXs, led us to attribute this peroxidatic activity to a Se‐dependent enzyme. Hemolymph GPX specific activity increases during development and a twofold stimulation was observed after an oxidative challenge with hemin, suggesting that enzyme synthesis is under regulatory control. A role for extracellular GPX as an antioxidant protection against oxidative damage produced by heme derived from digestion of blood hemoglobin is discussed. Arch. Insect Biochem. Physiol. 41:171–177, 1999. © 1999 Wiley‐Liss, Inc.     

Crystal structures of a poplar thioredoxin peroxidase that exhibits the structure of glutathione peroxidases: insights into redox-driven conformational changes

Glutathione peroxidases (GPXs) are a group of enzymes that regulate the levels of reactive oxygen species in cells and tissues, and protect them against oxidative damage. Contrary to most of their counterparts in animal cells, the higher plant GPX homologues identified so far possess cysteine instead of selenocysteine in their active site. Interestingly, the plant GPXs are not dependent on glutathione but rather on thioredoxin as their in vitro electron donor. We have determined the crystal structures of the reduced and oxidized form of Populus trichocarpaxdeltoides GPX5 (PtGPX5), using a selenomethionine derivative. PtGPX5 exhibits an overall structure similar to that of the known animal GPXs. PtGPX5 crystallized in the assumed physiological dimeric form, displaying a pseudo ten-stranded beta sheet core. Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. In addition, a computer model of a complex of PtGPX5 and its in vitro recycling partner thioredoxin h1 is proposed on the basis of the crystal packing of the oxidized form enzyme. A possible role of PtGPX5 as a heavy-metal sink is also discussed.     

Plant glutathione peroxidases

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H₂O₂ and its subsequent reduction to H₂O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known.     

The structure of NADH peroxidase from Streptococcus faecalis at 3.3 A resolution

NADH peroxidase (EC 1.11.1.1) previously isolated from Streptococcus faecalis 10C1 has been crystallized. The crystal structure has been solved by multiple isomorphous replacement and solvent-flattening at 3.3 A (1 A = 0.1 nm) resolution. The enzyme forms a tetramer consisting of 4 crystallographically related subunits. The monomer chain fold is in general similar to those of glutathione reductase and lipoamide dehydrogenase. FAD binds in the same region and in a similar conformation as in glutathione reductase. The unusual cysteine-sulfenic acid participating in catalysis is located at the isoalloxazine of FAD.     

Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities.

This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX). Both purified recombinant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione. Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate. Moreover, thioredoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective Vmax and Km values. We therefore conclude that these two plant GPX-like proteins are antioxidant enzymes showing PHGPX and TPX activities.     

Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase.

Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.     

NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803.

Here we isolated and characterized two genes (slr1171, slr1992) designated gpx-1 and gpx-2, respectively, encoding glutathione peroxidase (GPX)-like proteins (Gpx-1, Gpx-2) from Synechocystis PCC 6803. The deduced amino acid sequences for gpx-1 and gpx-2 showed high similarity to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. Surprisingly, both recombinant proteins in Escherichia coli were able to utilize NADPH, but not reduced glutathione, as an electron donor and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides as an acceptor. It seems accurate to refer to Gpx-1 and Gpx-2 as NADPH-dependent GPX-like proteins that serve as a new defense system for the reduction of unsaturated fatty acid hydroperoxides.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

Glutathione peroxidase activities from rat liver

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.     

Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Molecular and Catalytic Properties of Glutathione Peroxidase Family Proteins from Pinus tabulaeformis

Glutathione peroxidase (GPX) is one of the key enzymes that protect cells against oxidative damage caused by reactive oxygen species. Previous studies of plant GPXs focused mainly on angiosperms. In contrast, little information is available on the molecular characteristics of this gene family in gymnosperms. In this study, four GPX genes (PtaGPX1, 2, 3, and 4) were cloned from the gymnosperm Pinus tabulaeformis, which showed high protein sequence identity and similar expression patterns in various tissues. The four Pinus GPX proteins were expressed in Escherichia coli, and the purified proteins used thioredoxin, but not glutathione, as an electron donor. The four Pinus GPXs showed different enzymatic activities and kinetic characteristics, suggesting functional divergence. Two conserved Cys residues (corresponding to Cys44 and Cys92 of PtaGPX3) were identified in all plant GPXs, and their functions were assessed using site-directed mutagenesis. Cys44 and Cys92 of PtaGPX3 could form an intramolecular disulfide bond under oxidizing conditions. These two residues were critical components of active sites and contributed to catalytic activity. This study provides novel insights into the functional divergence and catalytic properties of the GPX family in gymnosperms.     

The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins

Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.     

Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution

The crystal structure of NADH peroxidase (EC 1.11.1.1) from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7% for all data in the resolution range 7 to 2.16 A. The standard deviations are 0.015 A in bond lengths and 3.0 degrees in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D2; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (His10) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO3H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.