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Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Grgic M,Williamson A,Kjæreng Bjerga GE,Altermark B,Leiros I

doi

10.1016/j.pep.2018.05.012

subject

Has Abstract

pub_date

2018-10-01 00:00:00

pages

100-108

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(18)30093-7

journal_volume

150

pub_type

杂志文章

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Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.