Deoxyribonucleotide synthesis ribonucleotide reductase

HU is an inhibitor of ribonucleotide reductase, a rate-limiting enzyme which catalyzes the conversion of ribonucleotides into deoxyribonucleotides. HU is thus a cytotoxic agent as it has the ability to inhibit DNA synthesis. Consequently, H U can affect only cells that are actively synthesizing DNA and, therefore, a drug of S-phase cell-cycle specific. Moreover, HU-mediated inhibition of ribonucleotide reductase is reversible, implying that the action of HU will exhibit a relatively straight forward concentration-time course dependence [2—4-]. 

Initial insight of the role of CSN in cell-cycle control came from the finding that csnl and csn2 deletion S. pomhe strains have an S-phase delay [52]. Interestingly, this effect did not occur in strains missing other CSN subunits. The S-phase delay was caused by the accumulation of the cell-cycle inhibitor Spdl (S-phase delayed 1), which is involved in the misregulation of the ribonucleotide reductase (RNR). RNR catalyzes the production of deoxyribonucleotides for DNA synthesis and... 

Ribonucleotide reductase is required for the formation of the deoxyribonucleotides for DNA synthesis. Figure 1-18-2 shows its role in dTMP synthesis, and Figure 1-18-3 shows all four nucleotide substrates ... 

Hydroxyurea (Hydrea) inhibits the enzyme ribonucleotide reductase and thus depletes intracellular pools of deoxyribonucleotides, resulting in a specific impairment of DNA synthesis. The drug therefore is an S-phase specific agent whose action results in an accumulation of cells in the late Gj- and early S-phases of the cell cycle. 

The nucleotides described thus far in this chapter all contain ribose (ribonucleotides). The nucleotides required for DNA synthesis, however, are 2 -deoxyribonucleotides, which are produced from ribonucleoside diphosphates by the enzyme ribonucleotide reductase. 

Ribonucleotide reductase is responsible for maintaining a balanced supply of the deoxyribonucleotides required for DNA synthesis. To achieve this, the regulation of the enzyme is complex. In addition to the single active site, there are two sites on the enzyme involved in regulating its activity (Figure 22.13). 

Ribonucleotide reductases are discussed in Chapter 16. Some are iron-tyrosinate enzymes while others depend upon vitamin B12, and reduction is at the nucleoside triphosphate level. Mammalian ribonucleotide reductase, which may be similar to that of E. coli, is regarded as an appropriate target for anticancer drugs. The enzyme is regulated by a complex set of feedback mechanisms, which apparently ensure that DNA precursors are synthesized only in amounts needed for DNA synthesis.273 Because an excess of one deoxyribonucleotide can inhibit reduction of all... 

Hydroxyurea interferes with the synthesis of both pyrimidine and purine nucleotides (see table 23.3). It interferes with the synthesis of deoxyribonucleotides by inhibiting ribonucleotide reductase of mammalian cells, an enzyme that is crucial and probably rate-limiting in the biosynthesis of DNA. It probably acts by disrupting the iron-tyrosyl radical structure at the active site of the reductase. Hydroxyurea is in clinical use as an anticancer agent. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

Many of the enzymes participating in de novo synthesis of deoxyribonucleotide triphosphates, as well as those responsible for interconversion of deoxyribonucleotides, increase in activity when cells prepare for DNA synthesis. The need for increased DNA synthesis occurs under three circumstances (1) when the cell proceeds from the G0, or resting, stage of the cell cycle to the S, or synthetic or replication, stage (fig. 23.26) (2) when it performs repair after extensive DNA damage and (3) after infection of quiescent cells with virus. When cells leave G0, for example, enzymes such as thymidylate synthase and ribonucleotide reductase, increase as well as the corresponding mRNAs. These increases in enzyme amount supplement allosteric controls that increase the activity of each enzyme molecule. Corresponding decreases in amounts of these enzymes and their mRNAs occur when DNA synthesis is completed. 

Reduction of ribonucleotides to deoxyribonucleotides, the essential first step in the synthesis of DNA, is catalyzed by the enzyme ribonucleotide reductase (equation 25). Three different types of ribonucleotide reductase are known, which differ in their requirement for metal, namely cobalt... 

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. 

Inhibition of nudeobase synthesis (2). Tet-rahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 274). The folate analogues aminopterin and methotrexate (amethopterin) inhibit enzyme activity. Cellular stores of THF are depleted. The effect of these antimetabolites can be reversed by administration of folinic acid (5-formyl-THF, leucovorin, citrovorum factor). Hydroxyurea (hydroxycarbamide) inhibits ribonucleotide reductase that normally converts ribonucleotides into deoxyribonucleotides subsequently used as DNA building blocks. 

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. 

Non-haem enzymes Ribonucleotide reductase dinuclear iron deoxyribonucleotide synthesis Tyrosine 3.2.1. 

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. 

Ribonucleotide reductase plays a critical role in the life cycle of all living organisms. By catalysing the conversion of ribonucleotides to deoxyribonucleotides, it holds a unique position at the biological crossroads between RNA synthesis and DNA synthesis. Its control of DNA synthesis and cell proliferation is mediated both by providing all precursors for replication and by keeping a balanced supply between them. Throughout the years, the metabolic key-role of RNR has been explored successfully in a variety of antiviral and antiproliferative therapies. 

The evolutionary transition from RNA to DNA is recapitulated in the biosynthesis of DNA in modem organisms. In all cases, the building blocks used in the synthesis of DNA are synthesized from the corresponding building blocks of RNA by the action of enzymes termed ribonucleotide reductases. These enzymes convert ribonucleotides (a base and phosphate groups linked to a ribose sugar) into deoxyribonucleotides (a base and phosphates linked to deoxyribose sugar). 

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. 

Ribonucleotide reductase is a cytoplasmic enzyme required by all growing cells. This enzyme converts ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA synthesis. The enzyme uses ribonucleotides in tiie diphosphate form, rather than the more familiar triphosphate form ... 

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. 

The Synthesis of Deoxyribonucleotides Is Controlled by the Regulation of Ribonucleotide Reductase... 

Regulation of ribonucleotide reductase is complex. The binding of dATP (deoxyadenosine triphosphate) to a regulatory site on the enzyme decreases catalytic activity. The binding of deoxyribonucleoside triphosphates to several other enzyme sites alters substrate specificity so that there are differential increases in the concentrations of each of the deoxyribonucleotides. This latter process balances the production of the 2 -deoxyribonucleotides required for cellular processes, especially that of DNA synthesis. 

The conversion of ribonucleotides into deoxyribonucleotides for the synthesis of DNA is key to the survival of any DNA-based life form. This process is completed by one of a variety of ribonucleotide reductases that reduce the furanose ring of a ribonucleic diphosphate acid monomer by replacing a hydroxyl functional group at the 2 -position with a hydrogen (Scheme 6) to generate the deoxyribonucleic acid... 

Synthesis of deoxythymidine nucleotides occurs differently from that of the other dNTPs, which are derived directly from a ribonucleotide reductase-catalyzed conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates (Figure 22.12). The terms thymidine and deoxythymidine (or dTTP and TIP) generally refer to the deoxyribonucleotide, because the ribonucleotide is not a normal metabolite. In the rare instances where the ribonucleotide of thymidine occurs, it is usually designated with an r preceding it, as in rTTP. 

See also Ribonucleotide Reductase and Deoxyribonucleotide Biosynthesis, Deoxyuridine Nucleotide Metabolism, Salvage Routes to Deoxyribonucleotide Synthesis, Nucleotide Analogs in Selection. 

Purine deoxyribonucleotides are derived primarily from the respective ribonucleotide (Fig. 6.2). Intracellular concentrations of deoxyribonucleotides are very low compared to ribonucleotides usually about 1% that of ribonucleotides. Synthesis of deoxyribonucleotides is by enzymatic reduction of ribonucleotide-diphosphates by ribonucleotide reductase. One enzyme catalyzes the conversion of both purine and pyrimidine ribonucleotides and is subject to a complex control mechanism in which an excess of one deoxyribonucleotide compound inhibits the reduction of other ribonucleotides. Whereas the levels of the other enzymes involved with purine and pyrimidine metabolism remain relatively constant through the cell cycle, ribonucleotide reductase level changes with the cell cycle. The concentration of ribonucleotide reductase is very low in the cell except during S-phase when DNA is synthesized. While enzymatic pathways, such as kinases, exist for the salvage of pre-existing deoxyribosyl compounds, nearly all cells depend on the reduction of ribonucleotides for their deoxyribonucleotide... 

The synthesis of deoxyuridine, cytidine, deoxycytidine and thymidine nucleotides from UMP (Fig. 6.13) involves three reactions CTP synthetase, ribonucleotide reductase, and thymidylate synthase (80). The first enzyme converts UTP into CTP and the second catalyzes the conversion of CDP, UDP, ADP and GDP into their respective deoxyribonucleotides. The last enzyme, thymidylate synthase, catalyzes the reductive methylation of deoxyUMP at the C-5 position giving deoxyTMP. The human enzyme has been extensively studied as it is a target enzyme in cancer chemotherapy. Besides these three enzymes, two other enzymes are involved in pyrimidine nucleotide synthesis and interconversion. DeoxyCMP deaminase converts deoxyCMP into deoxyUMP and deoxyUTP triphosphatase converts deoxyUTP into deoxyUMP. Giardia lamblia, and Trichomonas vaginalis lack both ribonucleotide reductase and thymidylate synthase and... 

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]. 

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