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Isolation of Methionine Sulfoxide Reductase A1 from the Psychrophile Colwellia psychrerythraea
Author(s) -
Bernhardson Andrew,
Schlessman Jamie L.,
Baumann Rick,
Domanski Tammy L.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a904-d
Subject(s) - psychrophile , methionine sulfoxide reductase , biochemistry , escherichia coli , thermostability , enzyme , biology , recombinant dna , methionine sulfoxide , methionine , gene , amino acid
Enzymes from psychrophilic organisms, whose optimal growth temperature is 5–10 °C, pose an intriguing kinetic problem, yet hold promise as catalysts in bioremediation and low‐temperature industrial applications. Preliminary studies, based on a few enzymes, suggest that psychrophilic organisms have evolved cold‐adapted proteins that overcome the kinetic barriers associated with low temperatures by displaying increased structural flexibility to enhance their catalytic efficiency. The recent publication of the Colwellia psychrerythraea genome provides an opportunity to produce recombinant enzymes from this psychrophilic bacterium. Studies of these enzymes will expand the psychrophilic protein database and enhance understanding of cold‐adaptation. Methionine sulfoxide reductase A1 (MsrA1), which restores function to some oxidatively‐damaged proteins, was selected as a test enzyme. Amino acid sequence alignment of MsrA1 with homologous mesophilic enzymes was performed to identify potential differences that may account for cold‐adaptation. The msrA1 gene was amplified from C. psychrerythraea genomic DNA and cloned into a plasmid. Recombinant MsrA1 protein was expressed in Escherichia coli and purified chromatographically. Thermostability studies will be performed using fluorescence spectroscopy, and enzymatic activity will be assessed using a protein repair assay.