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Studies of Psychrophilic Methionine Sulfoxide Reductases from Colwellia psychrerythraea
Author(s) -
Feagans Mika,
Cottrell Christopher A,
Domanski Tammy L,
Smith Virginia F,
Schlessman Jamie L
Publication year - 2008
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.22.1_supplement.758.4
Subject(s) - msra , methionine sulfoxide reductase , biochemistry , methionine sulfoxide , psychrophile , methionine , escherichia coli , chemistry , biology , gene , enzyme , amino acid
Methionine sulfoxide reductases (Msr) function as anti‐aging proteins by providing organisms with a mechanism to defend against the damaging effects of oxidation. These ubiquitous enzymes restore activity to oxidized methionine side chains in proteins. Methionine sulfoxide S and R epimers are repaired stereospecifically by MsrA and MsrB, respectively. Genomic analysis of the psychrophilic bacterium Colwellia psychrerythraea revealed five distinct msr genes, two msrAs , two msrBs and an msrA‐msrB fusion. Putative residues involved in substrate binding and reduction were identified by amino acid alignment of Cp MsrB proteins to mesophilic and hyperthermophilic homologs. In an effort to understand the significance of Msr function in this psychrophile, msrB1 was amplified from the Cp genome using PCR, ligated into a vector and transformed into competent E. coli host cells. Following confirmation of the gene sequence, the plasmid was transformed into expression host cells. Soluble recombinant Cp MsrB1 was produced as a histidine fusion and purified by affinity chromatography. Msr function was observed upon incubation with the peptide substrate H‐Lys‐Lys‐[Met(O)]‐Val‐Glu‐Asn‐Lys‐Lys‐OH at 4°C and monitored using MALDI‐TOF mass spectrometry. Temperature limitations of MsrB1 activity and conformational stability were assessed using MALDI‐TOF MS and CD and fluorescence spectroscopies.