Structurally dependent redox property of ribonucleotide reductase subunit p53R2.
p53R2 is a newly identified small subunit of ribonucleotide reductase (RR) and plays a key role in supplying precursors for DNA repair in a p53-dependent manner. Currently, we are studying the redox property, structure, and function of p53R2. In cell-free systems, p53R2 did not oxidize a reactive oxygen species (ROS) indicator carboxy-H2DCFDA, but another class I RR small subunit, hRRM2, did. Further studies showed that purified recombinant p53R2 protein has catalase activity, which breaks down H2O2. Overexpression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas overexpression of hRRM2 did not and resulted in a collapse of mitochondrial membrane potential. In a site-directed mutagenesis study, antioxidant activity was abrogated in p53R2 mutants Y331F, Y285F, Y49F, and Y241H, but not Y164F or Y164C. The fluorescence intensity in mutants oxidizing carboxy-H2DCFDA, in order from highest to lowest, was Y331F > Y285F > Y49F > Y241H > wild-type p53R2. This indicates that Y331, Y285, Y49, and Y241 in p53R2 are critical residues involved in scavenging ROS. Of interest, the ability to oxidize carboxy-H2DCFDA indicated by fluorescence intensity was negatively correlated with RR activity from wild-type p53R2, mutants Y331F, Y285F, and Y49F. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its fundamental enzymatic activity.
Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, California 91010, USA.
Lijun
L
Xue
Bingsen
B
Zhou
Xiyong
X
Liu
Tieli
T
Wang
Jennifer
J
Shih
Christina
C
Qi
Yvonne
Y
Heung
Yun
Y
Yen
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007624:KB Cells; D051336:Mitochondrial Membranes / Q000201:enzymology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018797:Cell Cycle Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; C405947:RRM2B protein, human; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit