1. Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation
- Author
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Jiqiang Ling, Sylvie Sinapah, Dieter Söll, and Sunna Helgadóttir
- Subjects
Models, Molecular ,Protein Conformation ,DNA Mutational Analysis ,Biophysics ,RNA, Transfer, Amino Acyl ,Biochemistry ,Article ,Amino Acyl-tRNA Synthetases ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,Genetics ,Protein biosynthesis ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,Conserved Sequence ,chemistry.chemical_classification ,Aminoacyl-tRNA ,Binding Sites ,ATP synthase ,biology ,Functional analysis ,Methanocaldococcus jannaschii ,Methanococcaceae ,Cell Biology ,biology.organism_classification ,SepCysS ,Enzyme ,chemistry ,Pyridoxal Phosphate ,Transfer RNA ,biology.protein ,Protein synthesis ,Archaea - Abstract
In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNACys to Cys-tRNACys. 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.
- Published
- 2011
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