RESUMO
Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3. GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%).
Assuntos
Glycine max/enzimologia , Glycine max/genética , Lactoilglutationa Liase/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Sequência Conservada , Dimerização , Biblioteca Gênica , Glutationa Transferase/metabolismo , Cinética , Lactoilglutationa Liase/química , Lactoilglutationa Liase/genética , Mamíferos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Sequences encoding proteins with homology to protein tyrosine phosphatases have been identified in Arabidopsis, soybean and pea. Each contains a predicted catalytic domain containing sequence motifs characteristic of tyrosine-specific protein phosphatases (PTPs) which play an important role in signal transduction in other eukaryotes and are distinct from dual-specificity, cdc25 or low-molecular-weight protein tyrosine phosphatases. Their identity as PTPs was confirmed by characterising the soybean PTP expressed as a recombinant His-tagged fusion protein. The enzyme had phosphatase activity towards p-nitrophenolphosphate (pNPP) and phosphotyrosine, but did not hydrolyse phosphoserine or phosphothreonine at a measureable rate. Phosphotyrosine containing peptides also served as substrates, with Km values in the micromolar range. Activity was abolished by inhibitors specific for tyrosine phosphatases (vanadate, dephostatin) but was unaffected by inhibitors of serine/threonine protein phosphatases (fluoride, cantharidin, metal-chelating agents). Gel filtration chromatography showed that the recombinant enzyme was a monomer. The Arabidopsis PTP sequence was isolated both as a genomic clone and as a partial EST, whereas the pea and soybean sequences were isolated as cDNAs. Southern analysis suggested a single gene in Arabidopsis and a small gene family in pea and soybean. In pea, PTP transcripts were present in embryos, and decreased in level with development; transcripts were also detectable in other tissues. The plant PTPs all contain a similar N-terminal domain which shows no similarity to any known protein sequence. This domain may be involved in PTP functions unique to plants.
Assuntos
Plantas/genética , Proteínas Tirosina Fosfatases/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , Southern Blotting , Domínio Catalítico , Sequência Conservada , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , Bases de Dados Factuais , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Pisum sativum/embriologia , Pisum sativum/enzimologia , Pisum sativum/genética , Plantas/embriologia , Plantas/enzimologia , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glycine max/enzimologia , Glycine max/genética , Distribuição TecidualRESUMO
An RT-PCR-derived clone encoding a stress-inducible glutathione transferase (GSTGm1) from soybean has been overexpressed in E. coli. The enzyme was active as the dimer GSTGm1-1 and showed GST and glutathione peroxidase activity toward diverse xenobiotics, including analogues of natural stress-metabolites. The selective herbicides, fomesafen and acifluorfen, were conjugated more actively with homoglutathione (hGSH), the major thiol in soybean, than with glutathione (GSH). No thiol preference was shown with the related herbicide, fluorodifen, while GSH was preferred with metolachlor and most non-herbicide substrates. Similar thiol-dependent specificities were observed in GST preparations from plants of varying GSH/hGSH content.
