Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.     

Expression of selenocysteine-containing glutathione S-transferase in eukaryote

Glutathione peroxidase (GPX) is a crucial antioxidant selenocysteine (Sec) containing enzyme which plays a significant role in protecting cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several methods have been used to generate GPX mimics, however, only a few of these methods involved genetic engineering and none of them have achieved specific site-directed incorporation of Sec without other modifications, which has hampered further structure-function studies. Here, we report for the first time the conversion of human glutathione transferase Zeta (hGSTZ1-1) into seleno-hGSTZ1-1 by means of genetic engineering in eukaryotes. Fluorescence microscopy images of the expression of Seleno-GST-green fluorescent protein chimaera indicated that we successfully achieved the read-through of the UGA codon to specifically incorporate Sec. Therefore, we achieved the conversion of human glutathione transferase Zeta (hGSTZ1-1) into a seleno-GST (seleno-hGSTZ1-1) by means of genetic engineering in eukaryotes. These results show that recombinant selenoproteins with incorporation of specific selenocysteine residues may be heterologously produced in eukaryotes by using a Sec insertion sequence in the 3' untranslated region (3'-UTR) of the mRNA, and the recombinant selenoproteins is single catalytically active residue and well-characterized structure. In this case a novel GPX activity of 2050±225 U/μmol was introduced into hGSTZ1-1 by substitution of serine 15 by Sec 15. This result will lay a foundation for preparing much smaller GPX mimics with higher activity.     

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.     

Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.     

Characterization of mitochondrial thioredoxin reductase from C. elegans

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.     

Three distinct-type glutathione S-transferases from Escherichia coli important for defense against oxidative stress

Previously, we characterized glutathione S-transferase (GST) B1-1 from Escherichia coli enzymologically and structurally. Besides GST B1-1, E. coli has seven genes that encode GST-like proteins, for which, except SspA, neither biological roles nor biochemical properties are known. Here we show that the GST-like YfcF and YfcG proteins have low but significant GSH-conjugating activity toward 1-chloro-2,4-dinitorobenzene and GSH-dependent peroxidase activity toward cumene hydroperoxide. Analysis involving site-directed mutagenesis suggested that Ser16 and Asn11 were important for the activities of YfcF and YfcG, respectively. On the contrary, no residue around the catalytic site of GST B1-1 has been demonstrated to be essential for catalytic activity. Deletions of the gst, yfcF, and yfcG genes each decreased the resistibility of the bacteria to hydrogen peroxide, which was recovered by transformation with the expression plasmid for the deleted enzyme. The inactive YfcF(S16G) and YfcG(N11A) mutants, however, could not rescue the knockout bacteria. Thus, E. coli has at least three GSTs of distinct classes, all of which are important for defense against oxidative stress in spite of the structural diversity. This seems consistent with the hypothesis that GSTs constitute a protein superfamily that has evolved from a thioredoxin-like ancestor in response to the development of oxidative stress.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Stress tolerance in transgenic tobacco seedlings that overexpress glutathione S-transferase/glutathione peroxidase

Overexpression of a tobacco glutathione S-transferase with glutathione peroxidase activity (GST/GPX) in transgenic tobacco (Nicotiana tabacum L.) enhanced seedling growth under a variety of stressful conditions. In addition to increased GST and GPX activity, transgenic GST/GPX-expressing (GST+) seedlings had elevated levels of monodehydroascorbate reductase activity. GST+ seedlings also contained higher levels of glutathione and ascorbate than wild-type seedlings and the glutathione pools were more oxidized. Thermal or salt-stress treatments that inhibited the growth of wild-type seedlings also caused increased levels of lipid peroxidation. These treatments had less effect on the growth of GST+ seedling growth and did not lead to increased lipid peroxidation. Stress-induced damage resulted in reduced metabolic activity in wild-type seedlings while GST+ seedlings maintained metabolic activity levels comparable to seedlings grown under control conditions. These results indicate that overexpression of GST/GPX in transgenic tobacco seedlings provides increased glutathione-dependent peroxide scavenging and alterations in glutathione and ascorbate metabolism that lead to reduced oxidative damage. We conclude that this protective effect is primarily responsible for the ability of GST+ seedlings to maintain growth under stressful conditions.     

Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp. W80

A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 ( C . W80) by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da. No potential signal peptide was found. The deduced amino acid sequence of the cDNA showed 40–63% and 37–46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. The C. W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site. However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX. Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H₂O₂ or phospholipid hydroperoxide. Transformed E. coli cells expressing the C . W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 m M paraquat treatments.
Accession number : The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.     

Cysteine is the presumed catalytic residue of Citrus sinensis phospholipid hydroperoxide glutathione peroxidase over‐expressed under salt stress

A citrus salt‐stress associated protein (Cit‐SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene ( csa ) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit‐SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit‐SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana

Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1-8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2-7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2-4.0 and 14.0-25.4 microM, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.     

Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3′-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3′-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3′-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.     

Selenocysteine insertion directed by the 3'-UTR SECIS element in Escherichia coli

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.     

A novel selenium-containing glutathione transferase zeta1-1, the activity of which surpasses the level of some native glutathione peroxidases

Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

谷胱甘肽过氧化物酶的硒代半胱氨酸插入元件

真核生物将硒代半胱氨酸插入蛋白质必需硒代半胱氨酸插入元件（ＳＥＣＩＳ）的参与,后者位于硒蛋白ｍＲＮＡ的３′非翻译区．采用ＲＮＡ折叠程序对１５个谷胱甘肽过氧化物酶基因进行计算机处理发现,其可能的ＳＥＣＩＳ中都具有３段保守碱基ＡＵＧＡ－Ａ（Ｇ）ＡＡ－ＧＡ．根据Ａ（Ｇ）ＡＡ位于顶环或者顶环上游５′臂的突环上,可将ＳＥＣＩＳ分为Ⅰ型和Ⅱ型结构