A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover

Abstract

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.

Keywords: metal ion–protein interaction; oligomerization; oxidation-reduction (redox); phylogenetics; ribonucleotide reductase; thioredoxin.

Figure 1.

Abundance and sequence of CRD were studied by phylogenetic and sequence analysis of NrdJ and NrdJd. A, maximum likelihood phylogenetic tree of a representative selection of NrdJ sequences. Subclasses with characterized members (NrdJm (green), NrdJd (red), and NrdJa+b (yellow)) and candidate subclasses (cyan, purple, orange, and light blue) are indicated with color. Lactobacillus leichmannii RNR (Ll) is located in subclass NrdJm, whereas the RNR from Thermotoga maritima (Tm) is not part of a defined subclass. B, appearance of the cysteine-rich domain mapped on a phylogenetic tree of a selection of nonmonomeric NrdJ sequences. The selected subset is marked by the dotted line in A. Subclasses with characterized members (NrdJd (red) and NrdJa+b (yellow)) and candidate subclasses (cyan, purple, orange, and light blue) are indicated with color. The presence of a CRD is indicated with a blue circle, absence with a white circle. The NrdJa+b subclass has the encoded CRD as a separate gene. C, logo of the CRD derived from 217 nonredundant sequences from subclasses NrdJd and unclassified NrdJ.

Figure 2.

Target enzymes from S. nassauensis are heterologously expressed and purified. Electrophoresis was performed with a 4–12% Bis-Tris Protein Gel. NrdJd-wt (Snas 4560): full-length NrdJd enzyme (1–947); NrdJdΔCRD: C-terminal truncation without CRD (1–714); CRD: CRD of NrdJd (715–947); glutaredoxin (Snas 1785); thioredoxin 1 (Snas 2647); thioredoxin 2 (Snas 6430); TR: thioredoxin reductase (Snas 6431).

Figure 3.

The presence of effector molecules modifies the activity profiles of NrdJd-wt and NrdJdΔCRD. A and B, four-substrate assay for NrdJd-wt (A) and NrdJdΔCRD (B) with or without effector. Each reaction contains the four potential substrates ADP, CDP, GDP, and UDP plus one of the potential effectors dATP, dCTP, dGTP, dTTP, or ATP. C and D, influence of the effector concentration on enzyme activity of NrdJd-wt (C) and NrdJdΔCRD (D) with one of the potential effectors dATP, dCTP, dGTP, dTTP, or ATP. Titrations were performed in a concentration range from 4 to 1000 μmol liter⁻¹, including measurements without effector. Data were obtained in three independent experiments. Error bars indicate the mean ± S.D.

Figure 4.

Effector molecules influence the oligomeric state of NrdJd. A and B, size exclusion chromatography of NrdJd-wt (A) and NrdJdΔCRD (B) with 1 mg ml⁻¹ protein each. C, GEMMA experiments with 0.075 mg ml⁻¹ NrdJd-wt and 100 μmol liter⁻¹ of one of the potential effectors. D, GEMMA experiment with 0.025 mg ml⁻¹ NrdJdΔCRD and 100 μmol liter⁻¹ of one of the potential effectors. E, GEMMA experiment with 0.075 mg ml⁻¹ NrdJd-wt and a titration of the dATP concentration. F, GEMMA experiment with 0.075 mg ml⁻¹ NrdJd-wt and a titration of the dGTP concentration. All GEMMA experiments represent five separate measurements for each condition.

Figure 5.

RNR activity of NrdJd-wt and NrdJdΔCRD depends on the applied reductant. A and B, enzyme activity of NrdJd-wt (red) and NrdJdΔCRD (green) in the presence of different concentrations of the reductants DTT (A) and TCEP (B). Assays were performed with CDP as substrate and dATP as effector. All experiments were performed in triplicate. Error bars indicate the mean ± S.D.

Figure 6.

A C-terminal zinc–binding site is elucidated by homology modeling and biochemically characterized. A, homology model of the last 30 amino acids of the CRD from S. nassauensis NrdJd, based on the crystal structure of tRNA(Ile2) 2-agmatinylcytidine synthetase (PDB ID: 4RVZ) (20). B, logo of the C-terminal end of S. nassauensis NrdJd-wt. C, activities with the reductant TCEP (narrow bars; black) and DTT (narrow bars; green) and zinc content (broad bars) of NrdJd-wt and the cysteine to alanine variants. The colors red and blue refer to the predicted location of the respective cysteine residue. Data were obtained in three independent experiments. Error bars indicate the mean ± S.D.

Figure 7.

Electron transfer between subunits is observed in mutant mixtures of NrdJd. A, specific activity per monomer of the C933A and C370A proteins tested individually and in an equimolar C933A/C370A mixture (mutant mix). B, relative activities of NrdJd-wt and the mutant mix in effector titrations with the substrate effector pairs CDP/dATP (100% equals k_cat = 15.3 min⁻¹) and GDP/dTTP (100% equals k_cat = 3.1 min⁻¹) and TCEP as reductant. C, activities of NrdJd-wt and the mutant mix with the substrates CDP or GDP and effector mixtures. TCEP was applied as reductant. Concentrations: dATP 250 μmol liter⁻¹, dTTP 1 mmol liter⁻¹. Data were obtained in three independent experiments. Error bars indicate the mean ± S.D.

Figure 8.

No electron transfer from potential native electron donor systems can be observed in activity measurements. A, specific activity of glutaredoxin and the thioredoxins in the presence or absence of NrdJd-wt (+RNR and −RNR), respectively. The assays were performed with or without additional oxidant used as substrate (+Ox or −Ox). The additional oxidants were insulin for the thioredoxins and HED for glutaredoxin. B, specific activity of NrdJd-wt in combination with glutaredoxin and the thioredoxins with the substrate CDP. C, activity assays with NrdJd-wt in the presence of cell lysate preparations from S. nassauensis with (green) or without (red) 250 μmol liter⁻¹ NADPH addition: protein-free lysate (Prep 1), cleared lysate (Prep 2), and complete lysate (Prep 3). No artificial reductant such as DTT was added. As positive and negative controls, RNR-assays with (+DTT) and without (−) the reductant DTT were performed. RNR activity in the preparations and controls was determined by HPLC measurement of CDP consumption and dCDP synthesis. All data were obtained in three independent experiments. Error bars indicate the mean ± S.D.