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M Fontecave

Publications and source records attributed to M Fontecave.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).

Aerobiosis↗
PubMed1992

Substitution of manganese for iron in ribonucleotide reductase from Escherichia coli. Spectroscopic and crystallographic characterization.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and two antiferromagnetically coupled oxo-bridged ferric ions. A refined structure of R2 has been recently obtained. R2 can be converted into apoR2 by chelating out the metal cofactor and scavenging the radical. This study shows that apoR2 has a very strong affinity for four stable Mn2+ ions. The manganese-containing form of R2, named Mn-R2, has been studied by EPR spectroscopy and x-ray crystallography. It contains two binuclear manganese clusters in which the two manganese ions occupy the natural iron-binding sites and are only bridged by carboxylates from glutamates 115 and 238. This in turn explains why the spin-exchange interaction between the two ions is very weak and why Mn-R2 is EPR active. Mn-R2 could provide a model for the native diferrous form of protein R2, and a detailed molecular mechanism for the reduction of the iron center of protein R2 is proposed.

Amino Acid Sequence↗
PubMed1992

8-Azidoadenosine and ribonucleotide reductase.

Inhibitors of ribonucleotide reductase are potential antiproliferative agents, since they deplete cells from DNA precursors. Substrate nucleoside analogues, carrying azido groups at the base moiety, are shown to have strong cytostatic properties, as measured by the inhibition of the incorporation of thymidine into DNA. One compound, 8-azidoadenosine, inhibits CDP reduction in cytosolic extracts from cancer cells. The corresponding diphosphate behaves as a substrate for ribonucleotide reductase while the triphosphate is an allosteric effector.

Adenosine↗
PubMed1992

Reduction of the small subunit of Escherichia coli ribonucleotide reductase by hydrazines and hydroxylamines.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and an antiferromagnetically coupled diferric center. Recent crystallographic studies [Nordlund, P., Eklund, H., & Sjöberg, B.-M. (1990) Nature 345, 593-598] have shown that both the radical and the diiron site are deeply buried inside the protein and thus strongly support the hypothesis of long-range electron-transfer processes within protein R2. This study shows that monosubstituted hydrazines and hydroxylamines are able to reduce the tyrosyl radical and the ferric ions, under anaerobic conditions. It allows characterization of the site from which those compounds transfer their electrons to the iron/radical center. The efficiency of any given reducing agent is not solely governed by its redox potential but also by its size, its charge, and its hydrophobicity. We suggest, as a possible alternative to the long-range electron-transfer hypothesis, that conformational flexibility of the polypeptide chain might exist in solution and allow small molecules to penetrate the protein and react with the iron/radical center. This study also shows that two reduction mechanisms are possible, depending on which center, the radical or the metal, is reduced first. Full reduction of protein R2 yields reduced R2, characterized by a normal tyrosine residue and a diferrous center. Both the radical and the diferric center are regenerated from reduced R2 by reaction with oxygen, while only the diferric center is formed by reaction with hydrogen peroxide.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.

Animals↗
PubMed1991

NAD(P)H oxidation by hydrogen peroxide in Escherichia coli.

A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity. This activity is compared to and found to be distinct from well-known E. Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.

Catalase↗
PubMed1991

Vitamin E derivatives as new potent inhibitors of microsomal lipid peroxidation.

Several synthetic Vitamin E derivatives are strong inhibitors of lipid peroxidation induced in rat liver microsomes either chemically by ferrous ions and ascorbate or enzymatically by NADPH and carbon tetrachloride. The relative activities of these inhibitors are consistent with their intrinsic antioxidant properties, as peroxyl radicals scavengers. Among them, a 3,4-dihydro-6-hydroxy-2H-1-naphtopyran with IC50 around 0.08 microM is one of the most potent yet known inhibitor of lipid peroxidation.

Animals↗
PubMed1990

Microsomal lipid peroxidation and oxy-radicals formation are induced by insoluble iron-containing minerals.

Lipid peroxidation, measured by malondialdehyde formation is induced in rat liver microsomes by insoluble iron-containing minerals (pyrite, magnetite, nemalite and an iron ore, minette de Lorraine) which are generally found either in iron mines or as contaminants of asbestos fibers. In spin-trapping studies using DMPO as a spin trap those minerals are also found to catalyze the oxidation of formate to carboxylate radicals by oxygen, via the formation of hydroxyl radicals. The two processes are mainly due to the presence of redox active iron at the surface of the solid particles and thus are greatly inhibited by desferrioxamine, a strong iron chelator. However, these reactions are not correlated.

Animals↗
PubMed1990

Human thioredoxin reactivity-structure/function relationship.

The reactivity of human thioredoxin (HTR) was tested in several reactions. HTR was as efficient as E. coli or plant and algal thioredoxins when assayed with E. coli ribonucleotide reductase or for the reduction of insulin. On the other hand, HTR was poorly reduced by NADPH and the E. coli flavoenzyme NADPH thioredoxin reductase as monitored in the DTNB reduction test. When reduced with dithiothreitol (DTT), HTR was much less efficient than thioredoxin m and thioredoxin f, the respective specific thioredoxins for the chloroplast enzymes NADP-malate dehydrogenase (NADP-MDH) and fructose 1,6 bisphosphatase (FBPase). Finally, HTR could be used in the photoactivation of NADP-MDH although less efficiently than thioredoxin m, proving nevertheless that it can be reduced by the iron sulfur enzyme ferredoxin thioredoxin reductase in the presence of photoreduced ferredoxin. Based on sequence comparisons, it was expected that HTR would display a reactivity similar to chloroplast thioredoxin f rather than to thioredoxin m. However the observed behavior of FTR did not exactly fit this prediction. The results are discussed in relation to the structural data available for the proteins.

Amino Acid Sequence↗
PubMed1990

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol.

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gräslund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.

Anaerobiosis↗
PubMed1990

High valent iron oxo intermediates might be involved during activation of ribonucleotide reductase: single oxygen atom donors generate the tyrosyl radical.

The active form of protein B2, the small subunit of ribonucleotide reductase from E. Coli, contains a binuclear non heme iron center and a tyrosyl radical. MetB2 is an inactive form that lacks the radical but retains the Fe(III) center. We earlier proposed that the function of the iron center was to catalyze the one-electron oxidation of the tyrosine residue from metB2 by dioxygen. We now report that incubation of metB2 with single oxygen atom donors, hydrogen peroxide, 3-chloroperoxybenzoic acid, monoperoxophtalate and 2-iodosobenzoate, also results in the formation of the tyrosyl radical, as monitored by UV-visible and EPR spectroscopy. A mechanism of reductive activation of dioxygen by the binuclear non heme iron center involving iron-oxo intermediates is proposed.

Chlorobenzoates↗
PubMed1990

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.

Anaerobiosis↗
PubMed1989

Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers.

The active form of protein B2, a homodimeric subunit of Escherichia coli ribonucleotide reductase, contains a diferric iron center and a cationic free radical localized to tyrosine 122 of one of the two polypeptide chains. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metB2 (earlier named B2/HU) is enzymatically inactive. Crude extracts of E. coli catalyze the interconversion of metB2 and B2. Radical introduction into metB2 requires a flavin reductase together with a second poorly defined protein fraction ("Fraction b") as well as dioxygen, NAD(P)H, and a flavin (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). We now find that ferrous ions can substitute for Fraction b and that the diferric center of metB2 is reduced during anaerobic incubation of the system with reduced flavin and ferrous ions. Spectroscopic evidence and isotope experiments suggest an in situ reduction of the diferric to a diferrous center. Admission of oxygen then results in the instantaneous oxidation of tyrosine 122 to the cationic radical coupled to the reformation of the diferric center, giving enzymatically active B2. These data suggest that reduced diferrous B2 is an intermediate between metB2 and B2 during radical introduction. In addition, we find that anaerobic incubation of B2 with reduced flavin results in the loss of the tyrosyl radical and the formation of metB2. This reaction occurs in the absence of Fraction b or ferrous ions. Our experiments reconstitute with defined reagents the interconversion between metB2 and B2 observed earlier in the E. coli extract. The flavin reductase system catalyzes the interconversion in both directions with dioxygen as the critical factor deciding whether activation or inactivation of ribonucleotide reductase occurs.

Animals↗
PubMed1989

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."

Aerobiosis↗
PubMed1987

The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase.

An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical. The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins. After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). The nature of the third protein, provisionally named Fraction b, is unknown. The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation. Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase. Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide. Hydrogen peroxide, formed by superoxide dismutase, is also harmful. In this case, catalase present in Fraction b might protect the system. Fraction b has, however, an additional unknown function in the overall process of radical generation.

Cyclic N-Oxides↗