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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
Published on: April 3, 2014
Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4
Miriam Grgic1, Adele Williamson1, Gro Elin Kjæreng Bjerga2
1The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT the Arctic University of Norway, N-9037, Tromsø, Norway.
Abstract:
Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.
Insights
Researchers characterized the ParI cytosine-specific DNA methyltransferase from Psychrobacter arcticus. While the enzyme shows thermal stability, its activity is labile after purification, suggesting horizontal gene transfer may explain its cold-adapted features.
Area of Science:
- Enzymology
- Molecular Biology
- Biochemistry
Background:
- Cytosine-specific DNA methyltransferases are crucial enzymes in prokaryotes, often part of type II restriction-modification systems.
- These enzymes play vital roles in gene expression, DNA replication, cell cycle, and DNA repair.
- DNA methyltransferases have significant biotechnological applications, including DNA mapping and epigenetic analysis.
Purpose of the Study:
- To characterize the ParI cytosine-specific DNA methyltransferase encoded by the parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4.
- To investigate the expression, purification, and enzymatic activity of recombinant ParI.
- To assess the thermal stability and potential lability of the purified enzyme.
Main Methods:
- Recombinant ParI was expressed and purified using N-terminal hexahistidine and maltose binding protein tags for solubility.
- Size exclusion chromatography was used to determine the monomeric state and molecular mass (54 kDa) of purified ParI.
- Thermal stability was assessed by melting temperature (53°C) and secondary structure analysis.
- Enzymatic activity was confirmed both in vivo and in vitro for recombinant and native ParI.
Main Results:
- Recombinant ParI was successfully expressed and purified, existing as a monomer with a molecular mass of 54 kDa.
- The protein exhibited a melting temperature of 53°C, with loss of secondary structure above 65°C.
- Both recombinant and native ParI demonstrated methyltransferase activity in vivo, and tagged recombinant ParI showed activity in vitro.
- A loss of in vitro activity was observed upon removal of solubility tags, indicating the active form is labile.
Conclusions:
- The ParI enzyme from Psychrobacter arcticus is a thermally stable protein, but its catalytic activity is sensitive to purification conditions.
- The observed lability of the active form suggests that ParI might be acquired through horizontal gene transfer, explaining its lack of typical cold-adapted features.
- Further studies are warranted to understand the precise mechanism of activity loss and the evolutionary implications of ParI's characteristics.
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