Substituting Selenocysteine for Catalytic Cysteine 41 Enhances Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione Peroxidase Expressed in Escherichia coli

Hazebrouck, Stéphane; Camoin, Luc; Faltin, Zehava; Strosberg, A. Donny; Eshdat, Yuval

doi:10.1074/jbc.m004985200

Cited by 78 publications

(49 citation statements)

References 37 publications

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“…E. coli lacks GPx-1 and PHGPx-like activities (25), and the negligible PHGPx activity that we detected in crude lysates from control cells transformed with empty vector was consistent with this. Cells overexpressing GPX1, GPX2, and GPX3 exhibited PHGPx-like activities that were ϳ4-, 8-, and 11-fold higher than background, respectively (Fig.…”

Section: The Peptide Sequences Encoded By Gpx1 Gpx2 and Gpx3supporting

confidence: 69%

Saccharomyces cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione Peroxidases

Avery

2001

Journal of Biological Chemistry

183

151

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The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1⌬, gpx2⌬, and gpx3⌬ deletion mutants; PHGPx activity was almost absent in a gpx1⌬/ gpx2⌬/gpx3⌬ triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1⌬/gpx2⌬/ gpx3⌬ mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H 2 O 2 . Unlike H 2 O 2 toxicity, delayed toxicity of 18:3 toward gpx1⌬/gpx2⌬/ gpx3⌬ cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with ␣-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.

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Section: The Peptide Sequences Encoded By Gpx1 Gpx2 and Gpx3supporting

confidence: 69%

Saccharomyces cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione Peroxidases

Avery

2001

Journal of Biological Chemistry

183

151

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“…It has been shown that fusion of SECIS elements to its appropriate location in mRNA of proteins to which a UGA codon was introduced in the open reading frame resulted in the incorporation of Sec. This has been shown both in mammalian proteins (51,52) and in bacteria (53). However, we could not identify a typical eukaryotic secondary structure in the 0.9-kb 3Ј-UTR sequence of Crgpx mRNA (Vienna RNA Package, version 1.4; Ref.…”

Section: Discussionmentioning

confidence: 61%

A Selenoprotein in the Plant Kingdom

Fu¹,

Wang²,

Eyal³

et al. 2002

Journal of Biological Chemistry

154

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Selenoproteins that contain the rare amino acid selenocysteine in their primary structure have been identified in diverse organisms such as viruses, bacteria, archea, and mammals, but so far not in yeast or plants. Among the most thoroughly investigated families of selenoenzymes are the animal glutathione peroxidases (GPXs). In the last few years, genes encoding GPX-like homologues from Chlamydomonas and higher plants have been isolated, but, unlike the animal ones, all of them have cysteine (rather than selenocysteine) residues in their catalytic site. In all organisms investigated that contain selenoproteins, selenocysteine is encoded by a UGA opal codon, which is usually a stop codon. We report here that, in Chlamydomonas reinhardtii, the cDNA-cloned sequence of a GPX homologue contains an internal TGA codon in frame to the ATG. Specific mRNA expression, protein production, and enzyme activity are selenium-dependent. Sequence analysis of the peptides produced by proteolytic digestion, performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirmed the presence of a selenocysteine residue at the predicted site and suggest its location in the mitochondria. Thus, our data present the first direct proof that a UGA opal codon is decoded in the plant kingdom to incorporate selenocysteine.

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“…Therefore, recombinant selenoprotein production is difficult, with the main barrier being a proper interaction between E. coli SelB and a given selenoprotein mRNA (12,13,21,43). Insertion of selenocysteine at a specific site into a protein can be achieved only by using a hairpin structure which strongly resembles the fdhF SECIS element at a proper distance downstream of a UGA codon (3,7,16,18). However, sequence requirements for the SECIS element would usually result in amino acid changes in the respective protein.…”

mentioning

confidence: 99%

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

Gursinsky

Gröbe

Schierhorn

et al. 2008

Appl Environ Microbiol

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Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the grampositive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of 75 Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNA Sec ) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of 75 Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.The 21st amino acid selenocysteine (Sec) is present in a variety of different eukaryotic, archaeal, and bacterial proteins (11,25,26,29,50). The insertion of selenocysteine into growing peptide chains is directed by an opal (UGA) codon (8, 51), which in the standard genetic code signals termination of translation. Selenoprotein synthesis in Escherichia coli has been elucidated and intensively studied by Böck and coworkers (6) and requires the products of the genes selA, selB, selC, and selD. selC encodes a selenocysteine-specific tRNA Sec that is first charged with serine by seryl-tRNA synthetase. It is converted to selenocysteyl-tRNA Sec by selenocysteine synthase, the gene product of selA. A low-molecular-weight selenium donor, selenophosphate, is required for this reaction. It is synthesized from ATP and selenide by selenophosphate synthetase, SelD (41).Recoding of UGA into selenocysteine requires a special mRNA motif, the selenocysteine insertion sequence (SECIS) (22). In mRNAs encoding bacterial selenoproteins, it is located at the 3Ј site of the UGA codon (52). This mRNA structure is recognized by the bacterial selenocysteine-specific elongation factor SelB, which functions homologously to the standard elongation factor EF-Tu (9, 24). SelB binds GTP and selenocysteyl-...

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Substituting Selenocysteine for Catalytic Cysteine 41 Enhances Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione Peroxidase Expressed in Escherichia coli

Cited by 78 publications

References 37 publications

Saccharomyces cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione Peroxidases

Saccharomyces cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione Peroxidases

A Selenoprotein in the Plant Kingdom

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

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