Ribonucleotide reductase, thioredoxin

Regeneration of reduced enzyme In order for ribonucleotide reductase to continue to produce deoxyribonucleotides, the disulfide bond created during the production of the 2 -deoxy carbon must be reduced. The source of the reducing equivalents is thioredoxin—a peptide coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two sulfhydryl groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, in the process forming a disulfide bond (see p. 19). 

The ribonucleotide reductase system, consisting of ribonucleotide reductase, thioredoxin and thioredoxin reductase, catalyzes the irreversible reduction of the four common ribonucleoside-5 -phosphates to the corresponding 2 -deoxyribonucleoside-5 -phosphates. This reaction provides the deoxy ribonucleotide precursors necessary for DNA synthesis. 

FIGURE 23.31 Conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, (a) The (—S—S—) / (—SH HS—) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH. (b) The structures of NDP and dNDP. 

E. coli differential centrifugation gel chromatography (M, = 2 x 10 ) DNA, phospholipids, ribonucleotide reductase, thioredoxin, thymidylate synthase, DNA polymerase 336... 

In the first step, thioredoxin reductase reduces a small redox protein, thioredoxin, via enzyme-bound FAD. This involves cleavage of a disulfide bond in thioredoxin. The resulting SH groups in turn reduce a catalytically active disulfide bond in nucleoside diphosphate reductase ( ribonucleotide reductase ). The free SH groups formed in this way are the actual electron donors for the reduction of ribonucleotide diphosphates. 

Ribonucleotide reductase (diphosphate) [EC 1.17.4.1], also known as ribonucleoside-diphosphate reductase, catalyzes the reaction of a 2 -deoxyribonucleoside diphosphate with oxidized thioredoxin and water to produce a ribonucleoside diphosphate and reduced thioredoxin. This system requires the presence of iron ions and ATP. Ribonucleotide reductase (triphosphate) [EC... 

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 22-41. The 3 -ribonu-cleotide radical formed in step (T) helps stabilize the cation formed at the 2 carbon after the loss of H20 (steps and (3)). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step ). Step (5) is the reverse of step ( ) regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step ). Ini , coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. 

All deoxyribonucleotides (used to synthesize DNA) are synthesized from ribonucleotides by the enzyme ribonucleotide reductase, which requires thioredoxin as a cofactor. This enzyme is highly regulated, for example, it is strongly inhibited by dATP—a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase deficiency (see below). 

The resulting thiol pair of the reduced thioredoxin is the reductant used for ribonucleotide reductase (Chapter 16). The standard redox potential E° of E. coli thioredoxin is -0.27 V, appropriately low for coupling to the NADPH / NADP+ system. 

Ribonucleotides are reduced to the 2 -deoxyribo-nucleotides (Eq. 16-21) that are needed for DNA synthesis by enzymes that act on either the di- or triphosphates of the purine and pyrimidine nucleosides348-351 (Chapter 25). These ribonucleotide reductases utilize either thioredoxin or glutaredoxin (Box 15-C) as the immediate hydrogen donors (Eq. 16-22). The pair of closely spaced -SH groups in the reduced thioredoxin or glutaredoxin are converted into a disulfide bridge at the same time that the 2 -OH of the ribonucleotide di- (or tri-) phosphate is converted to H20. While some organisms employ a vitamin B12-... 

Deoxyribonucleotides. A chain involving NADPH, a flavoprotein, thioredoxin, and ribonucleotide reductase converts either the ribonucleoside diphosphates or triphosphates to the corresponding 2-deoxy forms (step j, Fig. 25-14) as indicated in Eq. 25-16. 

Ribonucleotide reductase and the thioredoxin system. In some lactobacilli, vitamin B12 is involved in the reduction of ribonucleotide triphosphate to deoxyribonucleotide. 

The small protein thioredoxin supplies reducing equivalents to ribonucleotide reductase for the ribose ring reduction. Thioredoxin is itself reduced by another protein, thioredoxin reductase, a flavopro-tein. Reduced glutathione can also carry reducing equivalents to ribonucleotide reductase. In both cases, the ultimate source of reducing equivalents is NADPH. 

Thioredoxin is used by ribonucleotide reductase as reducing agent to reduce such diphosphonucleotides as ADP and UDP. NADPH is a substrate for thioredoxin reductase, and GTP is not used at all. 

The synthesis of DNA is dependent on a ready supply of deoxyribonucleotides. The substrates for these are the ribonucleoside diphosphates ADP, GDP, CDP, and UDP the enzyme responsible for the reduction of these substrates to their corresponding deoxy derivatives is ribonucleotide reductase, which has thioredoxin as a cosubstrate. 

The enzyme contains two catalytic sites, two regulatory sites and two specificity sites. The catalytic site binds the substrates, thioredoxin (reduced by NADPH + H+) and the nucleoside diphosphates. The allosteric regulatory site binds ATP as an activator in competition with dATP as an inhibitor. The specificity site binds dGTP, dTTP and dATP but not dCTP and modulates ribonucleotide reductase activity selectively for the four NDP substrates to balance the four dNTP pools. 

Glutaredoxin is another small ubiquitous protein with a different dithiol-active center which catalyzes GSH-disulfide transhydrogenase reactions. It is GSH-specific and cannot be reduced by thioredoxin reductase. It uses GSH and an NADPH-coupled glutaredoxin reductase to catalyze the reduction of a variety of disulfide substrates, including 2-hydroxyethyl-disulfide and ribonucleotide reductase [281]. Since GSSG inhibits the latter reaction, a high ratio of GSH to GSSG will promote the synthesis of deoxyribonucleotides, which is a likely control mechanism of DNA synthesis. 

Ribonucleotide reductase catalyses the reduction of the four common ribonucleotides to their corresponding deoxyribonucleotides, an essential step in DNA synthesis. All four ribonucleotides are reduced by the same enzyme [77], The enzyme (250 000 mol. wt.) is a complex of two proteins Mi which contains substrate and redox-active sulphydryl groups and M2 which contains both a (x-oxo-bridged binuclear iron centre (Fig. 5) [77] and a tyrosine moiety sidechain which exists as a free radical stabilised by the iron centre [78], This radical, which is only 5.3 A away from iron centre 1, has access to the substrate-binding pocket and is essential for enzyme activity. Electrons for the reduction reaction are supplied from NADPH via thioredoxin, a small redox-active protein. 

The metabolic function of the thioredoxin reductase-thioredoxin system is to supply reducing equivalents to a wide variety of acceptors. By far the best characterized of these is the E. coli ribonucleotide reductase system (23, 261) the reductase consists of two subunits, proteins B1 and B2 (262, 263), The B1 protein contains three reactive dithiol-disulfide pairs and appears to be the immediate acceptor of reducing equivalents from thioredoxin. As isolated, the three pairs are in the reduced state and, in the presence of the B2 protein, three molecules of ribonucleotide can be reduced prior to any input of reducing equivalents from thiore-... 

Deoxyribonucleotides, the precursors of DNA, are formed in E. coli by the reduction of ribonucleoside diphosphates. These conversions are catalyzed by ribonucleotide reductase. Electrons are transferred from NADPH to sulfhydryl groups at the active sites of this enzyme by thioredoxin or glutaredoxin. A tyrosyl free radical generated by an iron... 

Synthesis of Thymidine nucleotides first requires deoxyribonucleotide synthesis. The enzyme responsible for this step is Ribonucleotide Reductase. This enzyme acts on oxynucleotides in their diphosphate form. Thioredoxin, a small protein, is oxidized as the 2 hydroxyl group on the ribose ring is reduced. Oxidized Thioredoxin (S-S) is then reduced by FADH2 and NADPH. The products are the respective deoxynucleotide diphosphates which are further phosphorylated and then used for DNA synthesis. 

Regeneration of the ribonucleotide reductase is accomplished in Escherichia coli and in mammals by thioredoxin, a dithiol polypeptide (M.W. 12,000) coenzyme, which also plays a role in other protein disulfide reductase reactions. In thioredoxin, two cysteine residues in the sequence -Cys-Gly-Pro-Cys are converted to cystine. Reduced thioredoxin is regenerated by thioredoxin reductase, a flavoprotein enzyme that uses NADPH + H+. 

The reduction of ribonucleotides to deoxyribonucleotides is linked to NADPH by thioredoxin and thioredoxin reductase. Thioredoxin is a small protein which is oxidized from a dithiol to a disulfide form during ribonucleotide reduction. The dithiol form of thioredoxin is regenerated by NADPH and a specific flavoprotein, thioredoxin reductase. The thioredoxin system consisting of thioredoxin and thioredoxin reductase was first identified as the reducing system from E. coli by Reichard and co-workers (35, 36) and both proteins have since been purified to homogeneity (37, 38). 

Although the reduction of ribonucleotides to the corresponding 2 -deoxyribonucleotides is catalyzed by enzyme systems differing in their cofactor requirements and/or in the level of phosphorylation of the substrates, the overall reduction process is very similar in all systems. For all the systems NADPH is the ultimate reductant, the hydrogen is transferred by thioredoxin reductase to thioredoxin, which in turn is oxidized by ribonucleotide reductase with the concomitant production of a 2 -deoxyribonucleotide. In these systems the thioredoxin-thiore-doxin reductase reducing system can be replaced by dithiols such as dihydrolipoate or dithiothreitol. 

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