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Function

Ribonucleotide reductase (RNR) is an enzyme responsible for converting deoxyribonucleotides (dNTPs) from ribonucleotides (NTPs). It catalyzes this chemical reaction by removing the 2'-hydroxyl group of ribose ring of ribonucleotides. Therefore, this enzyme is also known as ribonucleoside diphosphate reductase (rNDP). In this way, this enzyme promotes the synthesis of precursors for regeneration and construction of DNA.

Because of its essential function for the structure of DNA, knowing that all cellular organisms have DNA as a hereditary structure, it is likely that RNR is present in all growing cells of all living beings. In addition, it is also speculated that the RNR played a key role in the transition from the "RNA world" to the "DNA world". In this question, RNR are subdivided into three classes, and the third class can function without oxygen and uses simple structures, thus, it is believed that this class is the oldest.

Classification and distribuition

RNRs can be classified into three distinct classes according to the method of generating thiyl ions triggered by the presence of a particular cofactor. RNRs I are characterized by having a complex quaternary structure and dependence on dioxygen to assemble the cysteine oxidant. RNRs I are present in viruses and in all domains of life and can be subdivided into RNR Ia, Ib, Ic, Id and Ie. RNRs Ia are the best characterized and will receive greater focus on this page. These enzymes present the diiron-tyrosyl radical and are composed of two types of subunits called R1 (or alpha) and R2 (or beta). The biggest difference between the Ia RNRs and the other class I RNRs is in the substitution of diiron-tyrosyl for other cofactors. For example, in RNR Ib, the two ferric ions are replaced by manganese. The cofactors present in the different class I RNRs are shown in the figure.

Although less studied, there are also class II and III RNRs, which are found only in microorganisms. Class II RNR has adenosylcobalamin, a vitamin B12 derivative, as a cofactor. Class III RNRs, on the other hand, use a [4Fe-4S]-activase to generate a stable glycyl radical capable of leading to the generation of the oxidant Cys at the active site of the RNR. The presence of RNRs of classes II and III is usually related to a better adaptation in environments with low oxygen availability, and RNRs III usually have their activity inhibited by oxygen. Complex eukaryotes, as they generally encode only RNR Ia, have low occupancy in hypoxic environments. Although they share an almost universal ribonucleotide reduction mechanism, the three classes of RNRs have low primary sequence similarity to each other, which may suggest independent emergence.

Several microorganisms are able to encode RNRs of different classes. In the case of Escherichia coli, RNR Ia is preferentially expressed in the growth phase under aerobic conditions, while RNR III is mainly expressed under anaerobic conditions. Furthermore, in environments with low iron availability or when there is biofilm formation, E. coli tends to preferentially express RNR Ib.

Geral structure of RNR Ia

Among the types of RNR, Ia is the best characterized, grouped among the class I RNRs, enzymes Oxygen dependent and found in all eukaryotic organisms and in some prokaryotes and viruses. The RNR class Ia is a structure that has the diiron-tyrosyl radical and is composed of two subunits, R1 (or alpha) and R2 (or beta). The large R1 structure, also known as alpha2-homodimer, encoded by the NrdA genes, contains the active site for nucleotide binding and reduction and two allosteric sites, involved in the allosteric regulation of substrate specificity and general enzyme activity. The small R2 structure, also known as beta2-homodimer, encoded by the Nrdb genes, contains a binding site for two iron ions in each polypeptide chain, where the interaction with the metallic cofactor occurs. Metal ion transitions are necessary cofactors to reduce nucleotides, as they generate an oxidant to an active cysteine ​​site (Cys), producing a thiyl radical that is necessary for the initiation of catalysis in all RNRs.

R1 subunit and active site

In Ia RNRs the catalytic site is located in the R1 subunit. This subunit is composed of two identical polypeptide chains, thus forming a homodimer. These chains are formed by both alpha helices and beta sheets, and close to the active site an alpha/beta barrel is formed, which is highly conserved among RNR classes. In addition to the active site, two important allosteric sites are also located in the R1 subunit: the specificity site (S site) and the activity site (A site).

The catalysis mechanism performed at the RNR active site is represented in the image.

Initially, the hydrogen bonded to the 3' carbon of the ribose ribonucleotide is transferred to the thiyl radical of Cys439. As a result, the 2'OH radical becomes more accessible for acid hydrolysis and its protonation is catalyzed by Cys225 and Glu441, resulting in the exit of a molecule from the substrate. After that, a proton is transferred from Cys462 to Cys225 which then transfers a hydrogen to the 2' carbon. Then, the transfer of a hydrogen to the 2' carbon of the substrate occurs concomitantly with the formation of a disulfide bridge between the Cys225 and Cys462 of the RNR. Finally, till radical regeneration occurs with the transfer of a hydrogen from Cys439 to the 3' carbon of the substrate, leading to the release of a deoxyribonucleotide. At the end of the catalysis, the disulfide bridge between Cys225 and Cys462 must still be reduced. The electron used in this reduction comes from a redox chain whose initial donor is NADPH. Numerous components participate in this redox chain, including Cys 754 and Cys759 from R1 itself. The reduction of the disulfide bridge between Cys225 and Cys462 would be very difficult to occur if there was a radical very close to the disulfide bridge, since the tendency would be for this radical to be reduced instead of the disulfide bridge. This is a likely explanation for the existence of a second subunit in the RNR, as the R2 subunit harbors a tyrosyl radical that is difficult to reduce.

R2 subunit and cofactor

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