Deoxyribonucleotide biosynthesis in green algae. Purification and characterization of ribonucleoside-diphosphate reductase from Scenedesmus obliquus

European Journal of Biochemistry

1993

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Thioredoxins and glutaredoxins, in their oxidized form, possess a single disulfide bridge located on an edge of the small compact molecules. In contrast to most other disulfide-containing proteins, this S-S bridge is cleaved by millimolar concentrations of sulfite in the absence of protein denaturing agents at pH 7-8 and ambient temperature; however, the reaction is not quantitative. Sulfitolysis of Escherichia coli thioredoxin was found to be associated .with an increase in fluorescence at 345 nm. A comparative study of sulfitolysis in 12 different thioredoxins and glutaredoxins of bacterial and plant origin has been made. Although they are all thought to be highly conserved in threedimensional structure, their reactivities towards sulfite and the effects of 6 M guanidinium chloride (not affecting, or enhancing sulfitolysis) vary strongly in the series, with E. coli thioredoxin being less reactive and plant thioredoxins and E. coli glutaredoxin being more susceptible molecules. Contrary to expectation, reaction with sulfite is not generally correlated with the presence of negatively or positively charged amino acid residues near the disulfide loop but is determined by individ-

Section: Discussionmentioning

confidence: 99%

Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin

Würfel

Häberlein

European Journal of Biochemistry

1993

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“…d ATP-Sepharose, successfully employed for purification of the algal enzyme [26], was not a good absorbant for …”

Section: (-)mentioning

confidence: 99%

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Willing

European Journal of Biochemistry

Auling

1988

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175

135

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAEcellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 ( M , =z 80000) and catalytic protein B2 (Mr = 100000, composed of two M , = 50000 polypeptides), which were both necessary for activity.In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (Z, o = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(II1)-containing pseudocatalase and of 0x0-bridged binuclear Mn(ll1) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(1l) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity.Manganese-deficient cell cultures were also grown in the presence of 54MnC12. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled.All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.

“…Ribonucleoside diphosphate reductase was purified from FdU-stimulated cultures of S. obliquus as described [15]. For improved purification of subunit U2, the pass-through fraction from dATP-Sepharose affinity chromatography was incubated at 55'C for 20 min, the precipitate removed by centrifugation, and the enzyme subunit was recovered from the supernatant by precipitation with ammonium sulfate (60% saturation).…”

Section: K 2 Materials and Methodsmentioning

confidence: 99%

“…An active yet short-lived enzyme was recently obtained from synchronous, or from fluorodeoxyuridinetreated cultures of the unicellular green alga, Scenedesmus obliquus [15]. Its inactivation by hydroxyurea and aerobic reactivation [16] suggested that the algal protein belongs to the same family of radical enzymes as found in E. co/i and animals cells.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the free radical in a plant ribonucleotide reductase

Harder

1987

FEBS Letters

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Ribonucleotide reductase of the green alga, Scenedesmus obliquus, contains an organic free radical in its catalytic subunit U2 which is quenched by hydroxyurea and regenerated upon aerobic incubation in the presence of Fe2+. The low‐temperature EPR spectrum exhibits a doublet centered at g = 2.0046. This EPR signal is indistinguishable from that of the tyrosine radical in mouse cell ribonucleotide reductase, indicating that the eukaryotes possess one common enzyme apparatus for deoxyribonucleotide biosynthesis.