Toward understanding phosphoseryl-tRNA ^Cys formation: The crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase

Abstract

A number of archaeal organisms generate Cys-tRNA Cys in a twostep pathway, first charging phosphoserine (Sep) onto tRNA Cys and subsequently converting it to Cys-tRNA Cys . We have determined, at 3.2-Å resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, ␣4 synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (␣␤) 2 phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.cysteine ͉ methanogen ͉ phosphoserine ͉ archaea ͉ aminoacyl-tRNA synthetase

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

“…An inherent structural asymmetry may also be a property of SepRS enzymes. The crystal structure of M. maripaludis SepRS, which crystallized with the full tetramer in the asymmetric unit, showed that the enzyme possesses only approximate 222 point group symmetry (9). The core regions of each monomer, which include the central canonical class II catalytic domain, obey the symmetry much more strictly than do several peripheral domains comprising the N terminus, C terminus, and an inserted domain not present in PheRS.…”

“…A crystal structure of the A. fulgidus enzyme with phosphoserine bound has also shown that recognition is accomplished, perhaps somewhat unexpectedly, by hydrogen bonding with polar but uncharged side chains, including Ser 231 , Ser 233 , and Asn 325 (8). Mutagenesis of the equivalent and conserved residues in M. maripaludis SepRS produces decreases of 10 3 -10 4 -fold in k cat /K m for activation of phosphoserine (9), suggesting that the crystal structure correctly identifies the site of amino acid binding.…”

“…In contrast, alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a homotetramer in solution (7). SepRS is encoded by a single gene (2), and crystal structures of the enzyme from Methanococcus maripaludis, Methanococcus jannaschii, and Archaeoglobus fulgidus reveal an ␣ 4 homotetramer in each case (8,9). Further, sedimentation equilibrium analyses of the M. jannaschii and A. fulgidus enzymes revealed that each exists as a tetramer in solution (8).…”

Ancient translation factor is essential for tRNA-dependent cysteine biosynthesis in methanogenic archaea

“…SepRS is a class II aaRS, and its structure consists of an α 4 -tetramer (9,10) that binds two tRNA Cys molecules in the crystal structure (10) and in solution (11). SepCysS has a similar structure to the pyridoxal-5′-phosphate-dependent Cys desulfurases (12).…”

Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation

“…Recently, some unique aaRSs were discovered that expand the code to noncanonical amino acids (2). Phosphoseryl-tRNA synthase (SepRS) attaches O-phosphoserine to tRNA Cys during the formation of Cys-tRNA in methanogens (3,4). Most excitingly, pyrrolysine (Pyl), the 22nd amino acid that is cotranslationally inserted in response to an inframe UAG codon, is charged to its unique amber suppressor tRNA (5) by pyrrolysyl-tRNA synthetase (PylRS) (6,7).…”