Studies with hydrogen isotopes on the mechanism of action of cobamide-dependent ribonucleotide reductase

Blakley, Raymond L.; Ghambeer, R. K.; Batterham, T. J.; Brownson, Carol A.

doi:10.1016/0006-291x(66)90176-8

Cited by 28 publications

(8 citation statements)

References 13 publications

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“…The concentration of HU used by them (0.20 M) is substantially higher than was found necessary to completely inhibit CDP reductase activity both in our experiments and in those of Elford (12). To demonstrate ribonucleotide reductase activity in E. coli extracts, it is necessary to use cell extracts with very high protein concentration (in our experiments, the protein concentration in cell extracts was always 20 mg/ml or higher) and a fairly large amount of radioactivity in the substrate (e.g., 5 x 105 counts per min of 3H-CDP in each assay). It is not clear why Rosenkranz et al failed to detect any inhibition of ribonucleotide reductase by HU; however, it might be pointed out that they used relatively low amounts of radioactive substrate ('4C-CDP; 6 x 103 counts/min).…”

Section: Discussionmentioning

confidence: 60%

Mechanism of Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli by Hydroxyurea

Sinha

Snustad

1972

J Bacteriol

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10 g [Difco]; tryptone, 13 g [Difco]; sodium chloride, 8 g; sodium citrate [dihydratel, 2 g; glucose, 1.3 g; distilled water, 1 liter), and EHA top-layer agar (agar, 6.5 g Pifco]; tryptone, 13 g Pifco]; sodium chloride, 8 g; sodium citrate [dihydrate], 2 g; glucose, 3 g; distilled water, 1 liter) (11) were used for growth of overnight bacterial cultures, dilutions, and plating, respectively. M9CA medium (M9 medium of Adams

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Section: Discussionmentioning

confidence: 60%

Mechanism of Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli by Hydroxyurea

Sinha

Snustad

1972

J Bacteriol

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“…The stereochemistry of the reduction of ribonucleoside triphosphates catalyzed by the reductase of L. leichmannii has been studied by techniques similar to those used for the E. coli enzyme. The results were identical, indicating that also the coenzyme Bu2-dependent ribonucleotide reductase from L. leichmannii catalyzes a stereospecific replacement of the 2'-OH group of the substrates with hydrogen (28,52,137,155,161). In the presence of reduced lipoic acid and a ribonucleoside triphosphate, the ribonucleoside triphosphate reductase catalyzed the transfer of tritium from 8H20 (in equilibrium with the hydrogens of the thiol groups of reduced lipoic acid) to the 5'position of the coenzyme and into the 2'-position of the deoxyribonucleoside triphosphate product, suggesting that coenzyme B12 functions as a hydrogen carrier during the reaction (6,185).…”

Section: Tides Ribonucleoside Diphosphate Reduction In E Colimentioning

confidence: 74%

Pyrimidine metabolism in microorganisms.

O’Donovan¹,

Neuhard²

1970

Bacteriological Reviews

304

180

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“…Our finding that hydrogen from formate is totally lost to the solvent during catalysis indicates that hydrogen is transferred to a site where it exchanges with the protons of water. Such an exchange occurs with class I and II enzymes and is there attributed to redox-active cysteine residues present in the enzymes as well as in thioredoxin and glutaredoxin (22,23). Also in the present case the exchange might occur on redox-active thiols of the reductase.…”

Section: Discussionmentioning

confidence: 95%

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

Mulliez

Ollagnier

Fontecave

et al. 1995

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

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During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical . The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence ofthe auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to C02, and isotope from [3H]formate appears in water.Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.Ribonucleotide reductases are a group of enzymes that provide the deoxyribonucleoside triphosphates required for DNA synthesis. Surprisingly, three different classes of reductases have been described, each with a distinct protein structure (1). At the substrate level, all enzymes apparently use identical radical chemistry for the reduction of C-2' of the ribose but employ widely different mechanisms to produce the required protein radical (2, 3). Class I enzymes, as exemplified by the aerobic Escherichia coli reductase, contain a diferric oxygenlinked iron center and a stable tyrosyl radical (4) that during the catalytic reaction generates a transient cysteinyl radical by intramolecular electron transfer (5, 6). Class II enzymes also generate a transient cysteinyl radical but employ adenosyl cobalamin for this purpose (7). Class III enzymes, for which the anaerobic E. coli reductase is the prototype, contain an iron-sulfur center and, in their active form, a glycyl radical (8,9). This radical is stable under anaerobic conditions, but it is oxygen sensitive, and class III enzymes only operate during anaerobiosis. The glycyl radical is generated from Sadenosylmethionine (10) by interaction of the reductase with a complex enzyme system present in E. coli, consisting of a 17.5-kDa iron protein ("activase") (11), flavodoxin (12), flavodoxin reductase (13), NADPH, and dithiothreitol.All reductases use their protein radical to generate an activated substrate radical (2, 3), which is subsequently reduced at C-2'. For the reduction, class I and class II enzymes employ enzyme-bound cysteine thiols (2,7,14,15), maintained in the reduced state by transthiolation with thioredoxin or glutaredoxin (16). For the E. coli class III reductase, the hydrogen donor has been unknown until now. The extreme oxygen sensitivity of the glycyl radical resulted in the loss of the radical during enzyme purification. This prevented studies of ribonucleotide reduction...

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Studies with hydrogen isotopes on the mechanism of action of cobamide-dependent ribonucleotide reductase

Cited by 28 publications

References 13 publications

Mechanism of Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli by Hydroxyurea

Mechanism of Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli by Hydroxyurea

Pyrimidine metabolism in microorganisms.

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

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