The Reducing Activity of Glutaredoxin 3 toward Cytoplasmic Substrate Proteins Is Restricted by Methionine 43

The proteins comprising the thioredoxin superfamily are found in large numbers in all organisms (1-3). These proteins use the redox chemistry of pairs of cysteines to carry out either reductive or oxidative reactions. The majority of these proteins present the simple basic structure of the thioredoxin fold along with a Cys-x-x-Cys active site. The prototypical member of this family is thioredoxin 1 of E. coli, which has a molecular weight of 12 kD. For many other members, the thioredoxin fold may be only one domain of a multi-domain protein or may have inserted into it polypeptide sequences that add to the functional activity of the protein (4-6). In E. coli, the basic thioredoxin structure is found in a number of cytoplasmic proteins that perform reductive reactions. These include thioredoxins 1 and 2, glutaredoxins 1, 2, 3, and 4 and NrdH (7-10). Excluding glutaredoxin 2, which has some features distinguishing it from the other proteins and has a molecular weight of 24.3 kD, these proteins range in molecular weight from 9.1 kD to 15.5 kD (2, 8). While some overlap in substrate specificity for these proteins has been observed, these proteins still can vary significantly in their specificity despite their quite similar three-dimensional structures and the short length of their polypeptide chains. The most important of these substrates for aerobic growth of E. coli is ribonucleotide reductase, the enzyme which generates deoxyribonucleotides essential for DNA replication. This enzyme, product of the nrdA,B genes, uses redox active cysteines for its reductive reaction and must be regenerated as an active enzyme by thioredoxin family members. Specifically, expression of any one of the three proteins, thioredoxin 1, thioredoxin 2 (when overexpressed) or glutaredoxin 1, is sufficient for regeneration of active ribonucleotide reductase (8, 11-14). A mutant lacking all three proteins is unable to grow and we have shown that ribonucleotide reductase is the only essential enzyme that requires the presence of at least one of these thioredoxin family members (15). We are interested in determining which features of the thioredoxin superfamily members are responsible for their differing specificities. Clearly for the glutaredoxins, a glutathione-binding site allows these proteins to receive their electrons from glutathione while thioredoxins are reduced by thioredoxin reductase. But this structural difference is unlikely to explain much or any of the variation in substrate specificity. Are the differences due to differing affinities between enzymes and substrates, to variations in redox potential among these proteins, to differences in the catalytic properties of their active site, or are there other unanticipated features of the proteins involved? To answer these questions, we have begun to analyze the differences in specificity between two of the E. coli thioredoxin family members, glutaredoxins 1 and 3. Glutaredoxin 3 is the most abundant of the three glutaredoxins, although, remarkably, the substrate of this protein has not yet been identified. Glutaredoxin 3 does not reduce ribonucleotide reductase efficiently and, therefore, by itself, does not generate enough activity of the enzyme in vivo to allow growth (2, 7). Glutaredoxin 2 is even less effective in this reductive reaction (7, 16). Thus, a mutant strain we have constructed (RO36), which is missing thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow on rich or minimal media (15). In addition to ribonucleotide reductase, these reductants are required for the regeneration of active phosphoadenylylsulfate (PAPS) reductase, an enzyme involved in sulfur assimilation and, thus the biosynthesis of cysteine (17). Our approach has been to use the properties of RO36 to isolate mutants of glutaredoxin 3, encoded by the grxC gene, that are able to reduce ribonucleotide reductase sufficiently to allow growth of E. coli on rich media (15). (RO36 also contains a null mutation in nrdH, which encodes a thioredoxin-like molecule capable of suppressing the growth defect when overproduced. (9)). In this way, we repeatedly obtained mutational alterations of grxC that affected only one amino acid of glutaredoxin 3, Met43, and changed it to either valine, leucine, or isoleucine. We also showed that these grxC mutations restore reduction of PAPS reductase, indicated by the ability of the cells to grow on minimal medium in the absence of cysteine. The three-dimensional structures of E. coli glutaredoxins 1 and 3 are quite similar (18-21). Superposition of the backbone atoms of 50 amino acids throughout the proteins gives a root-mean-square deviation of 1.78A signifying strong structural similarity. The two proteins share 33% amino acid sequence identity and contain the identical redox active site, Cys11-Pro12-Tyr13-Cys14, located at the beginning of α-helix 1 (Figure 1)(3, 22). The structures of both proteins consist of the core thioredoxin-fold, the N-terminal β1, α1, β2 motif and the C-terminal β3, β4, and α3 motif. The two motifs are connected by the loop that contains α2 (Figure 1)(3). Previous reports assigned redox potentials of -198 and -233mV for glutaredoxin 3 and glutaredoxin 1 respectively, indicating that glutaredoxin 1 is considerably more reducing than glutaredoxin 3 (23). One possible explanation for the increased activity of the glutaredoxin 3 mutants is that the amino acid substitutions have altered the redox potential of the protein or the reactivity of their active site cysteines so the protein behaves more like glutaredoxin 1. If that were the case, one might expect that Met43 would lie close to the active site of the protein. However, this residue is found some distance from the redox active site, located in the middle of α-helix 2, at a position equivalent to that of leucine 48 in glutaredoxin 1 (Figure 1). Leucine 48 is located only 2 positions downstream to residues of the binding site for RNR, which directs it to a disulfide between Cys754 and Cys759 located in the C-terminus of the R1 subunit of RNR. This proximity raised the possibility that the increased activity of the mutants resulted from an improved affinity for RNR. FIGURE 1 Structures of glutaredoxin 1 and 3. E. coli glutaredoxin 1 (Grx1, PDB file 1GRX) (right) and glutaredoxin 3 (Grx3, PDB file 3GRX) (left) consist of the core thioredoxin fold. The two structures are viewed from identical orientation. Secondary structures ... In this paper, we describe biochemical characterization and molecular dynamics simulations of the strongest of the mutant proteins, Met43Val. We also present further genetic selections for altered specificity mutants of Grx3. Our biochemical and molecular dynamics studies when compared with the wild-type Grx3 protein reveal a significant increase in the Vmax of the mutant protein towards its substrates, a slightly reduced redox potential, a decreased pKa of the amino-terminal cysteine of the active site, and an unchanged Km for substrate. These results, along with potential structural changes in the protein, are used to explain the increased efficiency of Grx3Met43Val.