Evolutionary relationships between yeast and bacterial homoserine dehydrogenases

Thomas, Dominique; Barbey, Régine; Surdin-Kerjan, Yolande

doi:10.1016/0014-5793(93)81359-8

Cited by 26 publications

(29 citation statements)

References 21 publications

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“…We finally investigated the ability of the CY306 strain to reveal critical residues such as cysteines involved in TRX interaction with its target. We took as an example the MET16 enzyme that contains two cysteine residues: C112, which does not possess any catalytic activity (46), and C245 in the consensus sequence KxECG(L͞I)H, which is required for MET16 activity (47). Before this work, the identity of the cysteine residue targeted by TRX in MET16 was unknown.…”

Section: Cy306 Trx1⌬ Trx2⌬ Yeast Strain As a Tool To Reveal Essentialmentioning

confidence: 99%

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

Vignols

Bréhélin

Surdin-Kerjan

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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All organisms contain thioredoxin (TRX), a regulatory thiol:disulfide protein that reduces disulfide bonds in target proteins. Unlike animals and yeast, plants contain numerous TRXs for which no function has been assigned in vivo. Recent in vitro proteomic approaches have opened the way to the identification of >100 TRX putative targets, but of which none of the numerous plant TRXs can be specifically associated. In contrast, in vivo methodologies, including classical yeast two-hybrid (Y2H) systems, failed to reveal the expected high number of TRX targets. Here, we developed a yeast strain named CY306 designed to identify TRX targets in vivo by a Y2H approach. CY306 contains a GAL4 reporter system but also carries deletions of endogenous genes encoding cytosolic TRXs (TRX1 and TRX2) that presumably compete with TRXs introduced as bait. We demonstrate here that, in the CY306 strain, yeast TRX1 and TRX2, as well as Arabidopsis TRX introduced as bait, interact with known TRX targets or putative partners such as yeast peroxiredoxins AHP1 and TSA1, whereas the same interactions cannot be detected in classical Y2H strains. Thanks to CY306, we also show that TRXs interact with the phosphoadenosine-5-phosphosulfate (PAPS) reductase MET16 through a conserved cysteine. Moreover, interactions visualized in CY306 are highly specific depending on the TRX and targets tested. CY306 constitutes a relevant genetic system to explore the TRX interactome in vivo and with high specificity, and opens new perspectives in the search for new TRX-interacting proteins by Y2H library screening in organisms with multiple TRXs. affinity chromatography ͉ gene disruption ͉ thioredoxin targets ͉ two-hybrid system ͉ redox T hioredoxins (TRXs) are small heat-stable oxidoreductases containing two redox-active half-cysteine residues in an active site with a conserved amino acid sequence CXXC (where X indicates various amino acids) (1). TRX was originally identified as hydrogen donor for the reduction of methionine sulfoxide (MetSO) (2) and of sulfate (3) in yeast and received its name when it was characterized as a small protein dithiol hydrogen donor to Escherichia coli ribonucleotide reductase (4). TRX is a hydrogen donor to peroxiredoxins (5) and is also required for a number of metabolic enzymes as part of their catalytic cycle. TRX is implicated in many cellular processes, including protein folding and regulation of transcription factors (6), protein repair after damage by oxidation, sulfur metabolism (7, 8), reduction of dehydroascorbate (9), germination, or reduction of the Calvin cycle and stromal enzymes in plants (10,11).Occurrence of TRXs in all genomes is highly variable and somewhat complex, depending on the organisms concerned. Most organisms, such as E. coli, the yeast Saccharomyces cerevisiae, and mammals (12) contain a limited number of TRX or TRX-like genes (usually fewer than five). In contrast, plants possess numerous TRX isoforms (13,14) or TRX-like proteins (11,(13)(14)(15). During the latest inventories in the Arabidopsis gen...

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Section: Cy306 Trx1⌬ Trx2⌬ Yeast Strain As a Tool To Reveal Essentialmentioning

confidence: 99%

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

Vignols

Bréhélin

Surdin-Kerjan

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…In these situations, electrons from GSH are generally transduced to their substrates through glutaredoxins. GSH may protect protein sulfhydryls from irreversible oxidation by glutathionylation (5,6). It also participates in the detoxification of peroxides, either directly or indirectly as a cofactor of glutathione peroxidase (1,7,8) and of several chemicals such as cadmium (9,10).…”

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confidence: 99%

A Genetic Investigation of the Essential Role of Glutathione

Spector

Labarre

Toledano

2001

Journal of Biological Chemistry

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In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of ⌬gsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of ⌬gsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that ␥-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a ⌬gsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions. Glutathione (GSH) is a broadly conserved tripeptide with a highly reactive thiol and a very low redox potential of about Ϫ240 to Ϫ250 mV. Its elevated concentration, up to 10 mM, and the fact that its reduced state is efficiently maintained by NADPH-dependent glutathione reductase (reviewed in Refs. 1-3) confers to this small molecule the properties of a cellular redox buffer. As a redox buffer, GSH is thought to be a major determinant in maintaining a reducing cellular thiol-disulfide balance. GSH is also important as an electron donor for several enzymes that have a reducing step in their catalytic cycle, such as ribonucleotide reductase (4). In these situations, electrons from GSH are generally transduced to their substrates through glutaredoxins. GSH may protect protein sulfhydryls from irreversible oxidation by glutathionylation (5, 6). It also participates in the detoxification of peroxides, either directly or indirectly as a cofactor of glutathione peroxidase (1,7,8) and of several chemicals such as cadmium (9, 10).In Saccharomyces cerevisiae and in probably all other GSHcontaining organisms, GSH is synthesized in two steps beginning with the action of ␥-glutamyl cysteine synthetase (GSH1) (11, 12), which catalyzes the condensation of glutamic acid to cysteine, in the rate-limiting step of this biosynthetic pathway. The product of this reaction is ␥-glutamylcysteine, which is combined with glycine through the action of glutathione synthase (GSH2) (13) to form GSH. Null mutations in GSH1 result in GSH auxotrophy (14 -16), demonstrating that GSH is essential in yeast. In mammals, GSH is also essential, as demonstrated by the embryonic lethality resulting from disruption of ␥-glutamyl cysteine synthetase ...

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“…3(a). These include the Nterminal end fingerprint region GXGXXG common to the vast majority of NAD(P)H binding sites (Wierenga et al, 1986) and the central consensus pattern for these enzymes (Thomas et al, 1993). Located 8 bp downstream from the TAA stop codon of hom lies the ATG start codon of thrC, an ORF encoding a protein of 356 aa.…”

Section: Streptomyces Coelicolormentioning

confidence: 99%

Characterization of the hom–thrC–thrB cluster in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331 The GenBank accession number for the sequence reported in this paper is AJ312095.

et al. 2002

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Three genes from the aminoethoxyvinylglycine (AVG)-producing Streptomyces sp. NRRL 5331 involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes appear to be organized in a bicistronic operon as deduced by disruption experiments. The thrB gene, however, is transcribed as a monocistronic transcript. The encoded proteins are quite similar to the HDH, HK and TS proteins from other bacterial species. The overall organization of these three genes, in the order hom-thrC-thrB, differs from that in other bacteria and is similar to that reported in the Streptomyces coelicolor genome sequence. This is the first time in which the gene cluster for the three last steps of threonine biosynthesis has been characterized from a streptomycete. Disruption of thrC indicated that threonine is not a direct precursor for AVG biosynthesis in Streptomyces sp. NRRL 5331 and suggested that the branching point of the aspartic acid-derived biosynthetic route of this metabolite should lie earlier on the threonine biosynthetic route.

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Evolutionary relationships between yeast and bacterial homoserine dehydrogenases

Cited by 26 publications

References 21 publications

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

A Genetic Investigation of the Essential Role of Glutathione

Characterization of the hom–thrC–thrB cluster in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331 The GenBank accession number for the sequence reported in this paper is AJ312095.

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