Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

Mulliez, Etienne; Ollagnier, Sandrine; Fontecave, Marc; Eliasson, R.; Reichard, Peter

doi:10.1073/pnas.92.19.8759

Cited by 89 publications

(97 citation statements)

References 26 publications

(25 reference statements)

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“…However, anaerobic conditions restore growth because of the presence of an E. coli alternative ribonucleotide reductase that is independent of disulfide reduction (35) and also by the likely decrease in non-native disulfide bond formation. In contrast, FIG.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

A Genetic Investigation of the Essential Role of Glutathione

Spector

Labarre

Toledano

2001

Journal of Biological Chemistry

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“…Class III enzymes use formate instead of a redoxin as an external reductant (44). With 3 H-formate as reductant, tritium is recovered in water rather than in the deoxyribonucleotide, demonstrating that the hydrogen of formate equilibrates with an enzyme-bound intermediate that exchanges protons with water.…”

Section: External Electron Donorsmentioning

confidence: 99%

“…These are either small proteins with redox-active thiols (thioredoxins or glutaredoxins) for class I and class II enzymes (42,43), or formate for class III enzymes (44). Class I, class II, and probably class III enzymes contain a redox-active cysteine pair for the direct reduction of the ribose moiety (45)(46)(47).…”

Section: Three Classes: An Overviewmentioning

confidence: 99%

Ribonucleotide Reductases

Jordan

Reichard

1998

Annu. Rev. Biochem.

662

639

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Ribonucleotide reductases provide the building blocks for DNA replication in all living cells. Three different classes of enzymes use protein free radicals to activate the substrate. Aerobic class I enzymes generate a tyrosyl radical with an ironoxygen center and dioxygen, class II enzymes employ adenosylcobalamin, and the anaerobic class III enzymes generate a glycyl radical from S-adenosylmethionine and an iron-sulfur cluster. The X-ray structure of the class I Escherichia coli enzyme, including forms that bind substrate and allosteric effectors, confirms previous models of catalytic and allosteric mechanisms. This structure suggests considerable mobility of the protein during catalysis and, together with experiments involving site-directed mutants, suggests a mechanism for radical transfer from one subunit to the other. Despite large differences between the classes, common catalytic and allosteric mechanisms, as well as retention of critical residues in the protein sequence, suggest a similar tertiary structure and a common origin during evolution. One puzzling aspect is that some organisms contain the genes for several different reductases. CONTENTS

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“…The reduction of the four ribonucleoside diphosphates (ADP, CDP, GDP and UDP) in Escherichia coli under aerobic conditions is catalyzed by ribonucleoside diphosphate reductase (RDPR, class I enzymes) through a mechanism involving free radicals generated on the sugar ring of the four natural substrates (Figures 1 and 3) [9][10][11][12][13][14][15]. A transient cysteinyl radical formed at the active site of the enzyme abstracts the 3'-hydrogen atom of the ribose and then a cascade of reactions involving two other cystein residues led to the reduction products, the 2'-deoxynuleotides dNDP (Figure 3) [14].…”

Section: Introductionmentioning

confidence: 99%

Key steps from the “RNA World” to the “DNA World”

Renard

Maurin

Chambert

et al. 2014

BIO Web of Conferences

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Abstract. In the « RNA World » hypothesis of the origin of life, RNAs are assumed to be the central macromolecules able to self-replicate, conserve information and catalyze the reactions necessary for a primitive metabolism and many enzymatic cofactors may be regarded as molecular fossils of the "RNA World". In the key steps involved in the transition from the RNA World to the DNA World, two main steps can be distinguished: (i) the synthesis of 2'-deoxyribonucleotides from ribonucleotides catalyzed nowadays by the enzyme ribonucleotide reductase and (ii) the synthesis of thymine, a base specific for DNA, from uracil which is a base specific for RNA, catalyzed today by the enzyme thymidylate synthase. In regard to the chemistry of sulfur used by both enzymes for achieving their respective catalysis, we were interested in the search for simple sulfur reactions able to catalyze such transformations and report here on first results in an approach from thionucleosides to the catalysis involved in the conversion of uracil to thymine. In the RNA World, the recruitment of cofactors was crucial to expand the catalytic repertoire of RNA and we also describe interesting preliminary results obtained in the prebiotic synthesis of pyridoxal (vitamin B6) that is the precursor of the key coenzyme pyridoxal phosphate (PLP) able to catalyze nowadays seven different enzymatic reactions.

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Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

Cited by 89 publications

References 26 publications

A Genetic Investigation of the Essential Role of Glutathione

A Genetic Investigation of the Essential Role of Glutathione

Ribonucleotide Reductases

Key steps from the “RNA World” to the “DNA World”

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