Abstract
Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.
Highlights
SepCysS catalyzes the conversion of tRNA-bound O-phosphoserine to Cys
The formation of disulfide and trisulfide linkages between Cys64 and Cys67 suggests that SepCysS employs both disulfide and persulfide intermediates for sulfur transfer
The requirement of multiple cysteines for sulfur transfer by SepCysS resembles the sulfur relay enzymes for tRNA thiolation, e.g. ThiI for 4-thiouridine biosynthesis [23, 24] and MnmA for 2-thiouridine biosynthesis [25, 26]. For both ThiI and MnmA, two cysteines are required for catalysis: one cysteine receives a sulfane sulfur from cysteine desulfurase and forms a persulfide enzyme adduct, and the other cysteine attacks the bridging sulfur of the persulfide to liberate the terminal sulfur and forms a disulfide with the first cysteine
Summary
SepCysS catalyzes the conversion of tRNA-bound O-phosphoserine to Cys. Results: Three Cys residues are essential for SepCysS activity, and two of them form disulfide and persulfide intermediates. Biochemical and mass spectrometric analysis demonstrated that Cys and Cys form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys272 increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates. SepCysS may mobilize sulfur from a sulfur source, form a persulfide group on a Cys residue, and donate the sulfur to Sep-tRNACys to generate Cys-tRNACys
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