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Mutational analysis of Sep‐tRNA:Cys‐tRNA synthase reveals critical residues for tRNA‐dependent cysteine formation
Author(s) -
Helgadóttir Sunna,
Sinapah Sylvie,
Söll Dieter,
Ling Jiqiang
Publication year - 2012
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2011.11.024
Subject(s) - transfer rna , biochemistry , cysteine , enzyme , biology , archaea , chemistry , rna , gene
In methanogenic archaea, Sep‐tRNA:Cys‐tRNA synthase (SepCysS) converts Sep‐tRNA Cys to Cys‐tRNA Cys . The mechanism of tRNA‐dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active‐site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.