The oxidative nuclease activity of human cytochrome c with mutations in Ω-loop C/D
Yu Feng1, Yao Dong1, Ke-Jie Du2
1School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
Abstract:
Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O2•-) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.
Insights
Engineered human cytochrome c (Cyt c) mutants exhibit enhanced DNA cleavage activity, particularly under acidic conditions. These findings highlight specific mutations
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Natural and artificial nucleases are crucial in biotechnology and biomedicine.
- Understanding protein DNA cleavage activity aids artificial nuclease design and DNA damage research.
Purpose of the Study:
- To engineer human cytochrome c (Cyt c) mutants with enhanced DNA cleavage activity.
- To investigate the role of specific mutations and reaction conditions on nuclease activity.
Main Methods:
- Engineering of four human Cyt c mutants (N52S, N52A, I81N, I81D).
- Assessing DNA cleavage and degradation activity compared to wild-type (WT) Cyt c.
- Mechanism and kinetic assays to determine the role of superoxide and peroxidase activity.
Main Results:
- Engineered Cyt c mutants displayed significantly enhanced DNA cleavage and degradation compared to WT Cyt c, especially under acidic conditions.
- Superoxide (O2•−) was identified as a key factor in the nuclease reaction.
- The I81D Cyt c mutant showed up to a 9-fold increase in peroxidase activity at pH 5.
Conclusions:
- Mutations at Ile81 and Asn52 in the Ω-loop C/D of Cyt c are critical for its nuclease activity.
- These findings suggest physiological relevance in DNA damage processes.
- Engineered Cyt c mutants hold potential for biomedical applications.
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