Biosynthesis and Degradation of Nucleotides:- Ribonucleotides Are the Precursors of Deoxyribonucleotides
Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides by direct reduction at the 2-carbon atom of the D-ribose to form the 2-deoxy derivative. For example, adenosine diphosphate (ADP) is reduced to 2-deoxyadenosine diphosphate (dADP), and GDP is reduced to dGDP. This reaction is somewhat unusual in that the reduction occurs at a nonactivated carbon; no closely analogous chemical reactions are known. The reaction is catalyzed by ribonucleotide reductase, best characterized in E. coli, in which its substrates are ribonucleoside diphosphates. The reduction of the D-ribose portion of a ribonucleoside diphosphate to 2-deoxy-D-ribose requires a pair of hydrogen atoms, which are ultimately donated by NADPH via an intermediate hydrogen-carrying protein, thioredoxin. This ubiquitous protein serves a similar redox function in photosynthesis and other processes. Thioredoxin has pairs of OSH groups that carry hydrogen atoms from NADPH to the ribonucleoside diphosphate. Its oxidized (disulfide) form is reduced by NADPH in a reaction catalyzed by thioredoxin reductase (Fig. 1), and reduced thioredoxin is then used by ribonucleotide reductase to reduce the nucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs). A second source of reducing equivalents for ribonucleotide reductase is glutathione (GSH). Glutathione serves as the reductant for a protein closely related to thioredoxin,
FIGURE 1 Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. Electrons are transmitted (blue arrows) to the enzyme from NADPH by (a) glutaredoxin or (b) thioredoxin. The sulfide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH; GSSG indicates oxidized glutathione). Note that thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group.
glutaredoxin, which then transfers the reducing power to ribonucleotide reductase (Fig. 1). Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in bio chemical transformations, once thought to be rare in biological systems. The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2 (Fig. 2). The R1 subunit contains two kinds of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the R1 and R2 subunits. At each active site, R1 con tributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical. The R2 subunit also has a binuclear iron (Fe3) cofactor that helps generate and stabilize the tyrosyl radicals (Fig. 2). The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis. A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 3. The 3-ribonu cleotide radical formed in step 1 helps stabilize the cation formed at the 2carbon after the loss of H2O (steps 2 and 3). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step 4). Step 5 is the reverse of step 1, regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step 6). In E. coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. Four classes of ribonucleotide reductase have been reported. Their mechanisms (where known) generally conform to the scheme in Figure 3, but they differ in the identity of the group supplying the active-site radical and in the cofactors used to generate it. The E. coli en zyme (class I) requires oxygen to regenerate the tyrosyl radical if it is quenched, so this enzyme functions only in an aerobic environment. Class II enzymes, found in other microorganisms, have 5-deoxyadenosylcobalamin (see Box 17–2) rather than a binuclear iron center. Class III enzymes have evolved to function in an anaerobic environment. E. coli contains a separate class III ribonucleotide reductase when grown anaerobically; this enzyme contains an iron-sulfur cluster (structurally dis tinct from the binuclear iron center of the class I en zyme) and requires NADPH and S-adenosylmethionine for activity. It uses nucleoside triphosphates rather than nucleoside diphosphates as substrates. A class IV ribonucleotide reductase, containing a binuclear manganese center, has been reported in some microorganisms. The evolution of different classes of ribonucleotide reductase for production of DNA precursors in different environments reflects the importance of this reaction in nu cleotide metabolism.
FIGURE 2 Ribonucleotide reductase. (a) Subunit structure. The functions of the two regulatory sites are explained in Figure 4. Each active site contains two thiols and a group (OXH) that can be converted to an active-site radical; this group is probably the OSH of Cys439, which functions as a thiy lradical. (b) The R2 subunits of E. coli ribonucleotide reductase (PDB ID 1PFR). The Tyr residue that acts as the tyrosyl radical is shown in red; the binuclear iron center is orange. (c) The tyrosyl radical functions to generate the active-site radical (OX), which is used in the mechanism shown in Figure 3.
MECHANISM FIGURE 3 Proposed mechanism for ribonu cleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the R1 subunit; the active-site radical (OX) is on the R2 subunit and in E. coli is probably a thiyl radical of Cys439 (see Fig. 22–40). Steps 1 through 6 are described in the text.
FIGURE 4 Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site (right). The diagram indicates in hibition or stimulation of enzyme activity with the four different substrates. The pathway from dUDP to dTTP is described later.
Regulation of E. coli ribonucleotide reductase is unusual in that not only its activity but its substrate specificity is regulated by the binding of effector molecules. Each R1 subunit has two types of regulatory site (Fig.2). One type affects overall enzyme activity and binds either ATP, which activates the enzyme, or dATP, which inactivates it. The second type alters substrate specificity in response to the effector molecule— ATP, dATP, dTTP, or dGTP—that is bound there (Fig. 4). When ATP or dATP is bound, reduction of UDP and CDP is favored. When dTTP or dGTP is bound, reduction of GDP or ADP, respectively, is stimulated. The scheme is designed to provide a balanced pool of precursors for DNA synthesis. ATP is also a general activator for biosynthesis and ribonucleotide reduction. The presence of dATP in small amounts increases the reduction of pyrimidine nucleotides. An oversupply of the pyrimidine dNTPs is signaled by high levels of dTTP, which shifts the specificity to favor reduction of GDP. High levels of dGTP, in turn, shift the specificity to ADP reduction, and high levels of dATP shut the en zyme down. These effectors are thought to induce several distinct enzyme conformations with altered specificities.
