Ribonucleoside diphosphates reduction

REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS DEOXYRIBONUCLEOSIDE DIPHOSPHATES... 

Figure 34-5. Reduction of ribonucleoside diphosphates to 2 -deoxyribonucleoside diphosphates.

The deoxyribonucleotides, except for deoxythymidine nucleotide, are formed from the ribonucleotides by the action of an enzyme complex, which comprises two enzymes, ribonucleoside diphosphate reductase and thioredoxin reductase (Figure 20.11). The removal of a hydroxyl group in the ribose part of the molecule is a reduction reaction, which requires NADPH. This is generated in the pentose phosphate pathway. (Note, this pathway is important in proliferating cells not only for generation... 

Deoxyrihonucleotides are generally formed by reduction of ribonucleoside diphosphates. This involves a series of redox reactions in which NADP+ and FAD play a role (see Section 15.1.1), with a subsequent electron transport chain. DNA contains thymine rather than uracil, so thymidine triphosphate (dTTP) is a requirement. Methylation of dUMP to dTMP is a major route to thymine nucleotides, and is dependent upon N, A °-methylenetetrahydrofolate as the source of the methyl group (see Box 11.13). 

C. Formation of deoxyribonudeotides by reduction of the 2 -hydroxyl group of the ribose sugars on the ribonucleoside diphosphates ADP and GDP is catalyzed by ribonucleotide reductase (Figure 10-3). 

Ribonucleotide reductase ribonucleoside diphosphate reductase) is a multisubunit enzyme (two identical B1 subunits and two identical B2 subunits) that is specific for the reduction of nucleoside diphosphates (ADP, GDP, CDP, and UDP) to their deoxy-forms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2-hydroxyl group are two sulfhydryl groups on the enzyme itself, which, during the reaction, form a disulfide bond (Figure 22.12). 

Hydroxyurea suppresses DNA synthesis by inhibiting ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Hydroxyurea is used in chronic cases of granulocytic leukemia that are unresponsive to busulfan. In addition, it is used for acute lymphoblastic leukemia. Hydroxyurea may cause bone marrow depression. 

The control of ribonucleotide reductase activity is affected in the classic feedback fashion by cellular nucleotide concentrations. dATP inhibits the reduction of all four ribonucleoside diphosphates. dTTP inhibits the reduction of only CDP and UDP. ATP is the positive effector for the reduction of these two nucleotides, and both dTTP and dGTP stimulate the reduction of GDP and ADP. Hydroxyurea, an antitumor agent, inhibits ribonucleotide reductase, and this depletes the deoxyribonucleotide supply required for tumor DNA biosynthesis. 

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. 

Thelander, L., 1974, Reaction mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Oxidation-reduction-active disulfides in the B1 subunit. J. Biol. Chem. 249 4858n4862. 

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... 

We turn now to the synthesis of deoxyribonucleotides. These precursors of DNA arc formed by the reduction ot ribonucleotides specifically the 2 -hydroxyl group on the ribose moiety is replaced by a hydrogen atom. The substrates are ribonucleoside diphosphates, and the ultimate reduclant is NADPH. The enzyme ribonucleotide reductase is responsible for the reduction reaction for all four ribonucleotides. The ribonucleotide reductases of different organisms are a remarkably diverse set of enzymes. Yet detailed studies have revealed that they have a common reaction mechanism, and their three-dimensional structural features indicate that these enzymes are homologous. We will focus on the best understood of these enzymes, that of E. coli living aerobically. 

Figure 22.15 Reduction of a ribonucleoside diphosphate by rNDP reductase. 

DNA synthesis depends on a balanced supply of the four deoxyribonucleotides [1]. In all living organisms, with no exception so far, this is achieved by reduction of the corresponding ribonucleotides (substrates can be either ribonucleoside diphosphates NDP or ribonucleoside triphosphates NTP) by NADPH (Scheme 10-1), through a complex free radical chemistry. The substrate specificity is modulated by a sophisticated allosteric mechanism which makes it possible for a single protein to regulate the reduction of all four conunon ribonucleotides. This aspect will not be discussed here. Three well-characterized classes of ribonucleotide reductases (RNRs) have been described so far, which all are radical metalloenzymes [2-5]. 

Fig. 41.18. Reduction of ribose to deoxyribose. Reduction occurs at the nucleoside diphosphate level. A ribonucleoside diphosphate (NDP) is converted to a deoxyribonucle-oside diphosphate (dNDP). Thioredoxin is oxidized to a disulfide, which must be reduced for the reaction to continue producing dNDP. N = a nitrogenous base.

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. 

What is conversion of ribonucleotides to deoxyri-bonucleotides Deoxyribonucleotides for DNA synthesis are produced by the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. 

As shown in Fig. 1, the enzyme catalyzes the reduction of ribonucleoside diphosphates (Fig. lA) by dithiothreitol (Fig. IB). K values for CDP and DTT are 70 x Af and 20 mAf, respectively. The requirement for a dithiol suggests that, as for other class II RNRs, such as the extensively studied enzyme from Lactobacillus leichmannii, the hydrogen donor is very likely to be a dithiol protein such as thioredoxin or glutaredoxin. However, there is still no experimental evidence that an archaeal thioredoxin operates as an electron source for RNRs. The enzyme also requires AdoCbl for which a value of 1 pAf has been obtained (Fig. 1C). Finally, the reaction has an optimal temperature of 80° (Fig. ID), with very little activity at 30°. How AdoCbl resists such a high temperature and how the enzyme controls Co-C bond homolysis required for catalysis in thermophilic AdoCbl-dependent enzymes is an intriguing question. These properties are shared by other isolated thermophilic class II RNRs (Table II). 

The isolated reductase (above) was used in assays to direct further fractionation experiments which culminated in the isolation of the physiological reducing system this turned out to be a previously unreci ized hydrogen transport system. This system, which connects ribonucleotide reductase to the NADPH-NADP+ system, was found to be a two-component system con.sisting of a small sulfhydryl protein, thioredoxin, and a flavoprotein, thioredoxin reductase. Thioredoxin is the reductant which specifically interacts with the ribonucleotide reductase. In the presence of catalytic amounts of thioredoxin, the thioredoxin reductase will link NADPH with the reduction of ribonucleoside diphosphates as follows ... 

The effects of dATP are complex. Low concentrations of dATP stimulate reduction of pyrimidine ribonucleoside diphosphates, whereas at high concentrations, dATP becomes a potent, general inhibitor of nucleotide reduction it is also seen that low concentrations of dATP counteract the stimulatory effects of dTTP and dGTP. It has been concluded that dATP converts the enzyme into an inactive state. 

The partly purified reductase from the Novikoflf tumor is specific for ribonucleoside diphosphates, and a single enzyme reduces CDP, UDP, ADP, and GDP. In analogy with the E. coli enzyme, the tumor reductase appears to require nonheme iron, as indicated by stimulation of reductase activity with iron salts and inhibition with iron-chelating agents. The purified reductase requires either a reductant such as dihydrolipoate or dithiothreitol, or an NADP-linked hydrogen transport system the E. coli thioredoxin-thioredoxin reductase couple will link the tumor reductase with NADPH. There appears to be a thioredoxin-like hydrogen transport system in the tumor and in rat liver 32) that links nucleotide reduction with NADPH. 

The pathways of deoxyribonucleotide synthesis from ribonucleotides are summarized below. The trivial names of the enzymes of ribonucleotide reduction are as follows ribonucleoside diphosphate reductase ribonu-cleoside triphosphate reductase thioredoxin reductase. Enzyme Commission numbers and systematic names have not yet been assigned. 

Ribonucleotide Reductase. The ribonucleotide reductases catalyze the reduction of ribonucleoside-diphosphates (or triphosphates) to the corresponding 2 -deoxyribonucleoside-diphosphates (or triphosphates), processes of preeminent importance for the biosynthesis of DNA (see Table 2, entry 4) (65,86). A variety of metal-containing cofactors have been discovered in the ribonucleotide reductases investigated to date (eg, a binuclear iron center in the mammalian and in the E. coli ribonucleoside diphosphate reductase) and the oxidation of two protein thiols to a disulfide unit is indicated as the direct source of the two reduction equivalents. The reductase from Lactobacillus leichmanii employs coenzyme B12 as cofactor in its (normal) base-on form and acts on purine- or pyrimidine-based ribonucleoside-triphosphates. Its crystal structure reveals not only the arrangement of the bound corrinoid cofactor, but also how the enzyme is... 

Earlier data and those derived from these inhibition studies can be summarized in a model of ribonucleoside diphosphate reductase in which the active site is formed both from B1 and B2. It contains active dithiols contributed by Bl and a free radical contributed by B2. The active dithiols donate the electrons required for ribonucleotide reduction while participating in catalysis the function and nature of the free radical remain unknown. 

Both the pyrimidines and the purines are built up from small precursor molecules which are readily available in the metabolic pool (page 185). The free bases are not synthesized as such but, while being assembled, the partially constructed ring structure reacts with a special phosphorylated pentose known as PRPP (5-phosphoribosyl-l-pyrophosphate) and forms a ribonucleotide. The deoxyribonucleotides, with the exception of TMP which is formed by methylation of deoxyuridylate, are formed by reduction of the corresponding ribonucleoside diphosphate. The conversion is precisely controlled by allosteric effects which ensure that all four deoxyribonucleotides are available in amounts appropriate for nucleic acid synthesis. 

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