|Ganichkin OM, Xu XM, Carlson BA, Mix H, Hatfield DL, Gladyshev VN, Wahl MC.
|Structure and catalytic mechanism of eukaryotic selenocysteine synthase
|J Biol Chem
|In eukaryotes and archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA[Ser]Sec into selenocysteyl-tRNA[Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA[Ser]Sec synthesis activity, and determined its crystal structure at 1.65 A resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homo-tetramer. The N-terminus, which mediates tetramer formation, and a large insertion, which remodels the active site, set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP where it could repel substrates with a free alpha-carboxyl group, suggesting why SecS does not act on free O-phospho-L-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues, which are strictly conserved in SecS orthologs but variant in related enzymes, coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate-loop accommodates the gamma-phosphate moiety of O-phospho-L-seryl-tRNA[Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA[Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors we offer a detailed reaction mechanism for the enzyme.