Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 Å resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (α2)2 homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNAGln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNAGlu and Glu-tRNAGln. The Glu-tRNAGln is then converted to Gln-tRNAGln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNAGlu and tRNAGln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNAGlu/tRNAGln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNAGln complex reveals the structural determinants responsible for specific tRNAGln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine

Rab1a rescues the toxicity of PRAF3

The PRA1-superfamily member PRAF3 plays pivotal roles in membrane traffic as a GDI displacement factor via physical interaction with a variety of Rab proteins, as well as in the modulation of antioxidant glutathione through its interaction with EAAC1 (SLC1A1). Overproduction of PRAF3 is known to be toxic to the host cells, although the factors capable of cancelling the toxicity remained unknown. We here show that Rab1a can rescue the cytotoxicity caused by PRAF3 possibly by “positively” regulating ER-Golgi trafficking, cancelling the “negative” modulation by PRAF3. Our results illuminate the close physiological relationship between PRAF3 and Rab proteins

the evolution of Gln-tRNA Gln formation

glutamyl-tRNA synthetase: a missing link i

Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation. Nucleic Acids Res

and archaeal selenocysteine formatio