Detection and Characterization of Glutathione S-Transferase Activity in Rice EF-1ββ′γ and EF-1γ Expressed in Escherichia coli

Kobayashi, Satoru; Kidou, Shin‐ichiro; Ejiri, Shin-ichiro

doi:10.1006/bbrc.2001.5799

Cited by 36 publications

(32 citation statements)

References 25 publications

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“…The N-terminal domain 1 is homologous to GST enzymes and maintains many features of the catalytic apparatus of these proteins (49,50). 3 However, the overall level of conservation in domain 1 is intriguingly much lower than in domain 2 for which the high-resolution solution structure is described here.…”

Section: Discussionmentioning

confidence: 85%

Solution Structure of the 162 Residue C-terminal Domain of Human Elongation Factor 1Bγ

Vanwetswinkel

Kriek²,

Andersen

et al. 2003

Journal of Biological Chemistry

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The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1B␣ and eEF1B␤, two guanine nucleotide exchange factors, and eEF1B␥. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1B␥ has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel ␤-sheet surrounded by ␣-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly.Elongation factor 1 (eEF1) 1 plays a central role in peptide elongation during the process of eukaryotic protein synthesis (reviewed by Merrick and Nyborg, Ref. 1). This multisubunit complex consists of two functionally distinct parts. eEF1A catalyzes the GTP-dependent delivery of aminoacylated tRNAs to the acceptor site of the ribosome. The eEF1B complex acts as an exchange factor (GEF) and recycles the inactive eEF1A-GDP released from the ribosome to the active GTP-bound state by stimulating nucleotide exchange on eEF1A. In metazoans, eEF1B is composed of three subunits, namely eEF1B␣, eEF1B␤, and eEF1B␥. Both the eEF1B␣ and eEF1B␤ subunits promote in vitro nucleotide exchange reactions through a homologous C-terminal catalytic domain (2). The exact role of the third subunit, eEF1B␥, is unknown. Unlike eEF1A and eEF1B␣ (and eEF1B␤), which are functional homologues of EF-Tu and EF-Ts in bacteria, eEF1B␥ is unique to eukaryotes. In fungi, eEF1B contains only eEF1B␣ and eEF1B␥.Recent structural information has notably extended the understanding of the portion of the eukaryotic elongation cycle taking place away from the ribosome. After the initial solution structure of a catalytically active 91-residue GEF domain from human eEF1B␣ (3) paved the way, a fuller picture of the nucleotide exchange mechanism was provided by analysis of the crystal structures of yeast eEF1A bound to the corresponding catalytic fragment of its exchange factor eEF1B␣, both in the absence and the presence of guanine nucleotides (4, 5). However, in vivo, the situation is much more complex with eEF1 in higher eukaryotes occurring as an assembly of at least four subunits. Based on various biochemical data, several models have been proposed for the quaternary organization of the eEF1 complex (6 -10). Although presenting some discrepancies, all these models agree on the tight binding of eEF1B␣ (and to a le...

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Section: Discussionmentioning

confidence: 85%

Solution Structure of the 162 Residue C-terminal Domain of Human Elongation Factor 1Bγ

Vanwetswinkel

Kriek²,

Andersen

et al. 2003

Journal of Biological Chemistry

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“…These genes could possibly have a wide range of functions including protection from oxidative stress, controlling translation in response to oxidative stress [34] or regulating protein folding in a chaperone like manner [35]. Unlike the high affinity of ElfA for GSH-affinity media, the eEF1Bc subunits/eEF1B complexes identified by Kobayashi et al and Kamiie et al did not exhibit affinity for glutathioneaffinity media, although eEF1Bc subunits purified from rice and Bombyx mori were amenable to purification by GSH-affinity chromatography [36,37] and GST activity was present in the purified EF1B complex preparations. Identification of the complete enzymatic and functional repertoire of ElfA will have to await eEF1B complex studies in A. fumigatus.…”

Section: Discussionmentioning

confidence: 96%

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Carberry

Neville

Kavanagh

et al. 2006

Biochemical and Biophysical Research Communications

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Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bc), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bc class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass = 20 kDa; pI = 5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean ± standard deviation, n = 3) of 3.13 ± 0.27 and 3.43 ± 1.0 lmol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

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“…Recently, we obtained data showing the existence of GST activity in both EF-1bb?g puriˆed from rice embryos and in recombinant rice EF-1g expressed in E. coli. 176) Although the function of the GST in EF1g is unknown at present, it may be a sensor that signals the redox state of the cell to the protein synthesizing machinery, as translation is activated by reduced glutathione and inhibited by oxidized glutathione.…”

Section: Structure and Functions Of Ef-1 And Ef-1 Genesmentioning

confidence: 99%

Moonlighting Functions of Polypeptide Elongation Factor 1: From Actin Bundling to Zinc Finger Protein R1-Associated Nuclear Localization

Ejiri

2002

Bioscience, Biotechnology, and Biochemistry

192

168

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Detection and Characterization of Glutathione S-Transferase Activity in Rice EF-1ββ′γ and EF-1γ Expressed in Escherichia coli

Cited by 36 publications

References 25 publications

Solution Structure of the 162 Residue C-terminal Domain of Human Elongation Factor 1Bγ

Solution Structure of the 162 Residue C-terminal Domain of Human Elongation Factor 1Bγ

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Moonlighting Functions of Polypeptide Elongation Factor 1: From Actin Bundling to Zinc Finger Protein R1-Associated Nuclear Localization

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