Ribonucleotide reduction mechanism

T wo main classes of adenosylcobalamin-activated enzymes function by facilitating the homolytic scission of the Co-C5 to cob(II)alamin and the 5 -deoxyadenosyl radical. The resultant 5 -deoxyadenosyl radical initiates catalysis by abstraction of a hydrogen atom, either from a substrate in the case of the class of enzymes that catalyze radical isomerizations, or by abstraction of a hydrogen atom from Cys408-/3-SH in the active site of ribonucleotide reductase II. The resultant enzymatic thiyl radical initiates the reduction mechanism by abstraction of a hydrogen atom from the ribonucleotide substrate. We shall begin with the isomerization/elimination reactions of adenosylcobalamin. 

Despite the advances, little is known about the mechanism of ribonucleotide reduction by class III enzymes. However, it is very likely that the reaction proceeds by a radical mechanism, similar to that of class I and II enzymes (Scheme 10-2) [22, 53], As a matter of fact, the class III enzyme is also a radical enzyme with a glycyl radical absolutely required for activity. Furthermore, mechanism-based inhibitors of class I and II RNRs, such as nucleotide analogs carrying azido, chloro or fluoro groups at the 2 position, are also excellent inhibitors of class III enzymes [54]. 

Hydroxyurea blocks DNA synthesis in many organisms with only minor impact on RNA and protein synthesis. It was the first synthetic inhibitor of ribonucleotide reduction to be recognized and has since been frequently applied in mechanistic studies, in cell biology, and, to a lesser extent, in clinical medicine as an inhibitor of DNA replication and cell proliferation. The cellular aspects have been reviewed by Timson . Our present discussion centers on the molecular mechanism of action, and on the important question whether the many diverse effects observed in vivo can be traced back to one single cause this matter is afflicted with some confusion. 

No mechanistic studies have been carried out on thermophilic class II RNRs to date. However, ribonucleotide reduction is likely to proceed by a radical mechanism similar to that common to class IE. coli and class IIL. leichmannii enzymes. In the following, this mechanism is described, with numbers of the key aminoacid residues from the P furiosus RNR sequence (Fig. 3). 

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). 

These results have been explained by a mechanism which supposes that a hydride ion (H ) is generated and that a concerted reaction takes place in which the leaving group (OH ) dissociates from C-2, but only when the attacking species (H ) is present (7, W). Beichard has sug sted that the iron-containing B2 subunit of the E. colt ribonucleotide reductase may somehow provide the postulated hydride ion. In ribonucleotide reduction by the L. leichmannii system, 5 -deoxyadenosylcobalamin participates as a coenzyme and may also be involved in generating a hydride ion. 

As we have seen, the biosynthesis of deoxyribonucleotides in E. coli has been studied in considerable depth discoveries made with that system greatly facilitated the exploration of this process in other cell types. Ribonucleotide reduction in several kinds of animal cells is evidently accomplished by a mechanism similar to that found in E. coli, whereas in Lactobacillus leichmannii and in certain Rhizobium and Clostridium species (2S), the process of ribonucleotide reduction differs distinctively from that in E. coli in that B12 cofactors are involved. 

Tatum EL, Fred EB, Wood HG and Peterson WH (1936) Essential growth factors for propionic acid bacteria, n. Nature of the Neuberg precipitate fraction of potato replacement by ammonium sulfate or by certain amino acids. J Bacteriol 32 157-174 Taylor MJ and Richardson T (1979) Application of microbial enzymes in food systems and in biotechnology. Adv Appl Microbiol 25 7-35 Thelander L, Graslund A and Thelander M (1983) Continual presence of oxygen and iron required for mammalian ribonucleotide reduction possible regulation mechanism. Biochem Biophys Res Comm 110 859-865... 

Various studies have also foeused on probing the mechanism of the ribonucleotide reduction process [314-316]. Notably, a compound proposed to be an intermediate in the reduction process, 3 -keto-2 -deoxynucleotide, has recently been trapped and characterized by high-field EPR speetroseopy [317]. 

A component of the ribotide reductase complex of enzymes, protein Ba, has been shown to contain two non-heme iron atoms per mole (77). This enzyme plays a vital, albeit indirect, role in the synthesis of DNA. Curiously, the lactic acid bacteria do not employ iron for the reduction of the 2 hydroxyl group of ribonucleotides. In these organisms this role has been assumed by the cobalt-containing vitamin Bi2 coenzyme (18). The mechanism of the reaction has been studied and has been shown to procede with retention of configuration (19). 

Figure 13.4 A proposed mechanism for all three classes of ribonucleotide reductases. Classes I and II RNRs require an active site Glu residue and a pair of redox-active Cys. Class HI RNRs lack the Glu and one of the Cys, and use formate as the reductant. (From Stubbe etal., 2001. Copyright 2001, with permission from Elsevier.)...

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 also inhibits DNA synthesis by its action on the M2 subunit of ribonucleotide reductase, but in this case it is the reduction of the purine nucleoside diphosphates which is inhibited and the pool of dTTP rises slightly (Turner et al., 1966 Adams and Lindsay, 1967 Krakoff et al., 1968 Adams et al., 1971 Skoog and Bjursell, 1974 Thelander et al., 1984). What prevents the pool rising dramatically is not clear, but some mechanism comes into play to reduce turnover of the dTTP pool (Nicander and Reichard, 1985). Its action is most satisfactorily reversed by changing the medium for drug free medium. 

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. 

Fig. 5. Mechanism of action of dinuclear non-haem iron enzymes utilising ferryl intermediates. Mechanisms for ribonucleotide reductase and methane mono-oxygenase adapted from that of Que [72]. Compound I and compound II define intermediates at the same oxidation state as the equivalent peroxidase intermediate (see Fig. 2). X is an unknown group suggested to bridge between the two iron atoms and form a cation radical. The nature of the electron required for the reduction of ribonucleotide reductase compound II is not clear - it is possible that this intermediate can also oxidise tyrosine [72].

Contents A. S. Mildvan, C. M. Grisham The Role of Divalent Cations in the Mechanism of Enzyme Catalyzed Phosphoryl and Nucleotidyl Transfer Reactions. - H.P.C.Hogenkamp, G.N.Sando The Enzymatic Reduction of Ribonucleotides. - W. T. Oosterhuis The Electronic State of Iron in Some Natural Iron Compounds. Determination by Mossbauer and ESR Spectroscopy. - A. Trautwein Mossbauer Spectroscopy on Heme Proteins. 

RNRs catalyze the reduction of ribonucleotides to deoxyribonucleotides, which represents the first committed step in DNA biosynthesis and repair.These enzymes are therefore required for all known life forms. Three classes of RNRs have been identified, all of which turn out to be metalloenzymes. The so-called class I RNRs contain a diiron site (see Cobalt Bn Enzymes Coenzymes and Iron-Sulfur Proteins for the other two types of RNRs). As diagrammed in Figure 5, these enzymes generate first a tyrosyl radical proximal to the diiron site in the protein subunit labeled R2, and then a thiyl radical in an adjacent subunit (Rl) that ultimately abstracts a hydrogen atom from the ribonucleotide substrate. This controlled tyrosine/thiol radical transfer must occur over an estimated distance of 35 A, and a highly choreographed proton-coupled electron transfer (PCET) mechanism across intervening aromatic residues has been proposed. Perhaps, even more remarkably,... 

Deoxyribonucleotides Synthesized by the Reduction of Ribonucleotides Through a Radical Mechanism... 

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