Abstract: Objective: To investigate the function of the prokaryotic expression product of the peroxiredoxin 3 gene (OsPrx3) from Oryza sativa L. in inhibiting DNA oxidative damage in vitro. Methods: Molecular recombination was used to construct a prokaryotic expression vector for OsPrx3 from ‘Dahonggu’ red rice and its site-directed mutant OsPrx3mC51A. The target protein was expressed in Escherichia coli and its in vitro hydroxyl radical scavenging activity was determined. Using the supercoiled form (SF) of the pMD18 plasmid as the substrate, DNA nicking experiments were conducted to identify the function of OsPrx3 in inhibiting DNA oxidative damage. Results: OsPrx3 was successfully expressed in E. coli. The scavenging rate of hydroxyl radical by OsPrx3 at 1.0 mg/mL was nearly 40% after 2 h of reaction. The results of hydroxyl radical scavenging assay revealed that OsPrx3mC51A was almost completely inactivated, confirming that cysteine residue at position 51 was the key catalytic site of this enzyme activity. The DNA nicking experiments using the pMD18 plasmid at 0.06 μg/μL showed that OsPrx3 at low concentrations (0.06–0.18 μg/μL) inhibited oxidative damage to the target DNA in a concentration-dependent manner. The highest inhibition rate of 88.9% was found at an OsPrx3 concentration of 0.18 μg/μL, which was 1.7-fold higher than that at 0.06 μg/μL. At optimal OsPrx3 concentration of 0.15 μg/μL, the inhibition rate of DNA oxidative damage was 77.5%. When a SF-DNA/nicked DNA (NF-DNA) ratio of 1.0 was set as the redox equilibrium point, the inhibitory effect of OsPrx3 at the optimal concentration could be maintained for at least 180 min, and the SF-DNA/NF-DNA ratio in the system still reached 1.5 after 180 min. Taken together, the prokaryotically expressed OsPrx3 could potently inhibit DNA oxidative damage in vitro. This study provides an experimental basis for the discovery and utilization of beneficial protein resources from red rice.

Key words: peroxiredoxin; oxidative damage; DNA; red rice; aging