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.

Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

“…Additional 200 μL of Milli-Q water was added to the reaction mixture. The concentration of the deoxy-CDP product was measured as described previously16,38. All of the in-vitro RNR enzymatic reactions were performed with three replicates.…”

Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction

“…This could be a sign of adaptation to a genome of higher GC-content—close to 60% in TV compared with 25–52% in the Firmicutes’ genomes—not only by its coding sequence, but also by how substrate specificity of the enzyme is regulated. Like all RNRs studied so far—except for RNRs in some members of the Herpesviridae family (Averett et al, 1983)—allosteric regulation of substrate specificity is intact in TVNrdJm and comparable to other described RNRs (Table 1) (Eliasson et al, 1999; Larsson et al, 2004; Loderer et al, 2017; Rozman Grinberg et al, 2018; Sintchak et al, 2002).…”

“…Activity of TVNrdJm was tested with the artificial reductants DTT and TCEP. While DTT is able to reduce the active site cysteine pair directly, TCEP can only work via the C-terminal cysteine pair (Domkin & Chabes, 2014; Loderer et al, 2017). The NrdJm exhibited 33% higher activity with TCEP compared to DTT at their respective maxima (Fig.…”

Peer Review #3 of "Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction (v0.1)"

“…Activity of TVNrdJm was tested with the artificial reductants DTT and TCEP. While DTT is able to reduce the active site cysteine pair directly, TCEP can only work via the C-terminal cysteine pair (Domkin & Chabes 2014;Loderer et al 2017). The NrdJm exhibited 33% higher activity with TCEP compared to DTT at their respective maxima (Figure 3 A).…”