Reduction of ribonucleoside triphosphates

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

The adenosylcobalamin-dependent RTPR catalyzes the reduction of ribonucleoside triphosphates (NTPs) to deoxyribonucleoside triphosphates (dNTPs) for DNA biosynthesis according to Equation (18). 

Fig.20 Schematic illustration of the reduction of ribonucleoside triphosphates to 2 -des-oxyribonucleoside triphosphates, as catalyzed by ribonucleotide reductases [182]...

In the reduction of ribonucleoside triphosphates, the L. leichmannii enzyme is able to use as reductant, dihydrolipoate, dithiothreitol, or dithio-erythritol, the oxidized forms of which are cyclic disulfides it has been shown that for each mole of dihydrolipoate oxidized, a mole of ribonucleoside triphosphate is reduced. The physiological reductant for the L. leichmannii reductase is a two-component, hydrogen transport system analogous to the thioredoxin-thioredoxin reductase ssrstem of E. coli the two components of the L. leichmannii system have been purified and are generally similar to those of the E. coli system, although their molecular weights are distinctive 7). The thioredoxin-thioredoxin reductase pair from E. coli will serve the L. leichmannii reductase, although the converse is not true. 

Influence of Various Deoxyribonucleoside Triphosphates on Reduction of Ribonucleoside Triphosphates - ... 

Another reaction that depends on adenosylcobalamin is the reduction of ribonucleoside triphosphates to the corresponding 2 -deoxy compounds, the building blocks of deoxyribonucleic acids. Methylcobalamin is formed, e. g., in the methyl-ation of homocysteine to methionine with N -... 

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). 

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... 

A deoxyadenosylcobalamin-dependent ribonucleoside triphosphate reductase has been partially purified from cell free extracts of the extreme thermophile, Thermus X-l 14). The enzyme preparation catalyzed the reduction of GTP and CTP at comparable rates, while UTP and ATP were reduced at only one-tenth the rate of GTP reduction. Only the dithiols could serve as reducing substrates. The enzyme has a temperature optimum of 70°, and the allosteric regulation of the enzyme activity is also temperature-dependent. The reduction of ATP is specifically stimulated by dGTP only at a higher temperature. Maximum stimulation of ATP reduction is observed at approximately 75°, while no stimulation can be detected at 37°. The molecular weight determined by gel filtration was approximately 80,000 but no information about the subunit structure is yet available. 

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]. 

The ribonucleoside triphosphate reductase of L. leichmannii is an allosteric enzyme, the activity of which is modified in a complex manner by deoxyribonucleoside triphosphates 28, 29). Reduction of each of the four substrates is maximally stimulated by a particular deoxyribonucleoside triphosphate, which Beck 29) terms a prime effector. The data of Table 16-III illustrate the specific nature of the effector stimulation prime effectors are indicated by the italicized data. The effector-induced stimulation of reductase activity appears to be countered in particular ways by deoxyribonucleoside triphosphates for example, dTTP inhibits the dATP-acti-vated reduction of CTP. It has been speculated that these complicated positive and negative allosteric effects produced by nucleotides may constitute a mechanism for ensuring that surpluses or shortages in the production of deoxyribonucleotides do not occur in the cell 29). 

Components of the incubation mixtures were present in the following concentrations (mM) substrates (2.6), effectors (0.4), Mg (16), dihydrolipoate (30), and 5,6-di-methylbenzimidazolylcobamide coenzyme (0.004). The data are in millimicromoles of reduction product formed per 20 minutes per 0.64 /ig of purified ribonucleoside triphosphate reductase from L. leichmcmnii. 

The discoveries made with the E. coli system provided the basis for studies of ribonucleotide reduction in other microbial species and in animal cells. The mechanism of ribonucleotide reduction in rat tissues resembles that of E. coli in many ways, whereas ribonucleotide reduction in Lactobacillus leichmannii differs distinctively, in that coenzyme Bk takes part in a reduction accomplished at the nucleoside triphosphate level. Each of the three reductases that have been extensively purified reduces ribonucleotide substrates representing all four of the ribonucleosides of RNA and each displays remarkable allosteric regulatory properties (see below). 

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. 

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