Ribonucleoside Triphosphate Reductases

I. 17.4.2], also known as ribonucleoside-triphosphate reductase, catalyzes the reaction of a 2 -deoxyribonucleo-side triphosphate with oxidized thioredoxin and water to produce a ribonucleoside triphosphate and reduced thioredoxin. In this case, cobalt and ATP are cofactors. 

The best-studied enzymes to date that contain glycyl radicals are pyruvate formate-lyase (PFL) and a ribonucleoside triphosphate reductase (ARR), both isolated from anaerobically growing E. coli. These enzymes play central roles in the anaerobic metabolism of the bacterium. The first catalyzes the reversible formation of acetyl-CoA and formate from pyruvate and coenzyme A, while the second is responsible for synthesizing the deoxyribonucleotide monomers of the polymer DNA. It is intriguing to note that formate, a product of the PFL reaction, is a substrate for the ARR. It supplies the reducing equivalents needed for each round of deoxynucleotide synthesis. ... 

Lawrence, C. C., and Stubbe, J., 1998, The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from Lactobacillus leichmanrdi. Curr. Opin. Chem. Biol. 2 650n655. 

Licht, S. S., Booker, S., and Stubbe, J., 1999a, Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation. Biochemistry 38 12219 1233. 

Lawrence CC, Stubbe J. The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from... 

Chen D, Abend A, Stubbe J, Frey PA. Epimerization at carbon-5 of (5 R)-(5 - H)adenosylcobalamin by ribonucleoside triphosphate reductase cysteine 408-independent cleavage of the Co-C5 bond. Biochemistry 2003 42 4578 584. 

Ribonucleotide reductases with an absolute requirement for adenosyl-cobalamin (Fig. 1) as a coenzyme have been demonstrated only in microorganisms (7). These reductases may be classified into two groups based on the nature of the nucleotide substrate utilized ribonucleoside triphosphate reductases, which act upon the ribonucleoside triphosphates... 

Ribonucleoside Triphosphate Reductase from Euglena gracilis... 

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. 

Ribonucleoside Triphosphate Reductase from Pithomyces chartarum... 

G. Role of Adenosylcobalamin in Ribonucleoside-Triphosphate Reductase Reactions.. 335... 

It was demonstrated that these three enzymes have not only deoxyribosyl transferase, but also deoxynucleoside kinase, ribonucleoside triphosphate reductase, adenosine deaminase and DNA polymerase activity. 

Metal ions are clearly essential for the ribonucleoside triphosphate reductase isolated from the filamentous cyanophyte, Anabaena 7119, one of the many blue-green algae that depend on deoxyadenosylcobalamin for deoxyribonucleotide synthesis The purified enzyme possesses a molecular weight of 72,000 (estimated by gel filtration) with no subunit structure. It does not reduce ribonucleotides in the absence of divalent cations Ca" " is most effective but Mg" " and Mn" also support enzyme catalysis. Judging from their optimum concentration (5-10 mM) the metal ions are not only necessary to complex the substrate triphosphate but should have an effect on the enzyme protein itself. 

An efficient double oxidation of D-xylose to D-gf/ycero-aldopentos-2,3-diulose (2,3-diketo-D-xylose, 45) has been achieved (80% yield) using pyranose dehydrogenase from the mushroom Agaricus bisporus (Scheme 9). The gram scale 2 -deoxygenation of ATP has been achieved using a recombinant ribonucleoside triphosphate reductase from Lactobacillus leichmannii ... 

Fig. 2. Hypothetical role of cobalamine coenzyme (DBC) in the action of ribonucleoside triphosphate reductase.

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. 

Brunella, A. and Ghisalba, O. (2000) Recombinant Lactobacillus leichmannii ribonucleoside triphosphate reductase as biocatalyst in the preparative synthesis of 2 -deoxyribo-nucleoside-5 -triphosphates. f Mol. Catal. B, 10,215-222. 

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