title: Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.
abstract: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Sunna
authors.initials: S
authors.lastname: Helgadóttir
authors.forename: Sylvie
authors.initials: S
authors.lastname: Sinapah
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Jiqiang
authors.initials: J
authors.lastname: Ling
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D017016:Methanococcaceae / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
chemical_list: D012346:RNA, Transfer, Amino Acyl; D011732:Pyridoxal Phosphate; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine