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Escherichia coli Orf135 (NudG) mutant protein specific for oxidized dATP
1Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Abstract:
Damaged 2'-deoxyribonucleotides cause mutations, cancer, cell death, and aging. The Escherichia coli Orf135 (NudG) protein catalyzes the hydrolysis of various 2'-deoxyribonucleotides including an oxidized form of dATP, 2-oxo-1,2-dihydro-2'-deoxyadenosine 5'-triphosphate (dAOTP, 2-hydroxy-2'-deoxyadenosine 5'-triphosphate). The best substrate is 5-methyl-2'-deoxycytidine 5'-triphosphate (dCmTP), and the protein prefers dCmTP over dAOTP by ∼200-fold in vitro. To make the enzyme specific for the mutagenic nucleotide dAOTP, a double mutant protein (E33A plus D118E) was designed and produced in E. coli. The purified mutant protein showed one order of magnitude higher dAOTP preference over dCmTP. The split protein based on this mutant may potentially be used to detect dAOTP in living cells.
Insights
Researchers engineered a bacterial protein to specifically detect a damaging DNA building block, 2-oxo-1,2-dihydro-2'-deoxyadenosine 5'-triphosphate (dAOTP). This engineered protein shows a higher preference for dAOTP, potentially aiding in cellular damage detection.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Damaged 2'-deoxyribonucleotides contribute to mutations, cancer, cell death, and aging.
- The *Escherichia coli* Orf135 (NudG) protein hydrolyzes various 2'-deoxyribonucleotides, including the oxidized form of dATP, 2-oxo-1,2-dihydro-2 '-deoxyadenosine 5 '-triphosphate (dAOTP).
- The native Orf135 protein exhibits a higher affinity for 5-methyl-2 '-deoxycytidine 5 '-triphosphate (dCmTP) than for dAOTP (approximately 200-fold preference in vitro).
Purpose of the Study:
- To engineer a mutant *E. coli* Orf135 (NudG) protein with enhanced specificity for the mutagenic dAOTP.
- To investigate the substrate preference of the engineered mutant protein for dAOTP over dCmTP.
- To explore the potential application of the engineered protein in detecting dAOTP within living cells.
Main Methods:
- Site-directed mutagenesis was employed to create a double mutant protein (E33A plus D118E) from the *E. coli* Orf135 (NudG) protein.
- The mutant protein was expressed and purified from *E. coli*.
- Enzyme kinetics and substrate specificity assays were performed in vitro to compare the preference of the mutant protein for dAOTP versus dCmTP.
Main Results:
- The double mutant protein (E33A plus D118E) was successfully produced in *E. coli*.
- The purified mutant protein demonstrated a significantly altered substrate preference, exhibiting a one order of magnitude higher preference for dAOTP compared to dCmTP.
- This enhanced specificity indicates a shift in the enzyme's catalytic activity towards the mutagenic nucleotide.
Conclusions:
- The engineered double mutant protein exhibits increased specificity for dAOTP, a mutagenic nucleotide.
- This modified protein holds potential for developing biosensors or diagnostic tools to detect dAOTP in biological systems.
- The development of specific probes for damaged nucleotides could advance research into aging, cancer, and mutation prevention.
