Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR), the rate-limiting enzyme in DNA synthesis, catalyzes reduction of the different ribonucleotides to their corresponding deoxyribonucleotides. The crucial role of RNR in DNA synthesis has made it an important target for the development of antiviral and anticancer drugs. Taking account of the recent developments in this field of research, this review focuses on the role of thioredoxin and glutaredoxin systems in the redox reactions of the RNR catalysis.

Keywords: DNA synthesis; Glutaredoxin; Replication; Ribonucleotide reductase; Thiol disulfides; Thioredoxin.

Figure 1

Crystal structure of Class I aerobic ribonucleotide reductase complex (A), and proposed reaction mechanism of ribonucleotide reductase catalysis (B). A: This is based on the crystal structures of the R1 and R2 proteins (Protein Data Bank ID: 1RLR and 1RIB). The figure shows the presence of substrate in R1 subunit and dinuclear iron center in R2 subunit. The ribonucleotide reductase complex (RNR) complex is a tetramer with the dimer of R1 subunit and the dimer of R2 subunit. The allosteric regulatory domain of R1 subunit (ATP-cone) binds either ATP or dATP to regulate the enzymatic activity (adapted from Logan et al[9]); B: The figure describes the reduction of nucleoside diphosphate (NDP) to deoxyribonucleoside diphosphates (dNDP) by class I RNR (E. coli). The reduction is initiated by a thiyl radical (Cys 439) by abstracting the 3′-hydrogen from the NDP. A water molecule is lost and the two cysteines (Cys 225 and Cys 462) then deliver the required reducing equivalents, generating a 3′-ketodeoxynucleotide which is subsequently reduced to give dNDP (adapted from Holmgren et al[4]).

Figure 2

Subunit organization of ribonucleotide reductase complex. Amino acids are shown with E. coli numbering which are crucial for the radical transfer and ribonucleotide reductase (RNR) catalysis. The R2 subunit contains the iron-oxygen cluster (Fe-O-Fe) which reacts with dioxygen to generate a stable tyrosyl radical in Tyr 122 required for the RNR catalysis. The radical transfer pathway from Tyr 122 to the active-site Cys 439 in R1 subunit involves the network of Asp 84, His 118, Asp 237, Trp 48, Tyr 356 in R2 subunit and Tyr 730, Tyr 731 in R1 subunit[2-5]. The Cys 225, Cys 462, Asn 437 and Glu 441 residues are involved in binding the substrate nucleoside diphosphate (NDP) in R1 subunit. During the catalysis, the disulfide bond between Cys 225 and Cys 462 is reduced by the C-terminal shuttle dithiols[2-5]. The figure is adapted and modified from Holmgren et al[4].

Figure 3

The mechanistic model for the role of thioredoxins and glutaredoxins for the ribonucleotide reductase catalysis. After the completion of one turnover cycle of ribonucleotide reductase (RNR) catalysis, a disulfide bond is formed between the conserved cysteine pair at the active site (shown in the circle). Shuttle dithiol function present at the C-terminal CXXC motif of the neighboring subunit reduces the disulfide bond through disulfide-exchange. Then, the resulting disulfide bond at the C-terminal tail is reduced by the thioredoxin/glutaredoxin (Trx/Grx) systems resulting in an active R1 to continue the next cycle of RNR catalysis. The Grx system can also reduce the C-terminal thiols by the glutathionylation mechanism[4,25,26]. For simplicity, only the reduction of active site of one subunit by the C-terminal shuttle dithiols of the neighboring subunit is shown in the diagram. The figure is adapted and modified from Holmgren et al[4].

Figure 4

Activity profile of mouse ribonucleotide reductase in the presence of the thioredoxin and glutaredoxins system. Mouse R1 (120 μg/mL) and R2 (40 μg/mL) were assayed with dithiothreitol, thioredoxin (Trx) and glutaredoxin (Grx) systems. The Trx system contained 3.6 μmol/L Trx1, NADPH and TrxR. The Grx system contained 1 μmol/L Grx1, 4 mmol/L glutathione , NADPH and glutathione reductase. Combinations of 3.6 μmol/L Trx1 or the whole Trx system with the Grx system were also monitored (data adapted from Zahedi Avval et al[26]).