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The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated K_m values for serine and ATP, accompanied by decreases in k_cat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3"-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. termophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.

The activities of two nuclearly encoded seryl-tRNA synthetases (SerRS) are required in different protein-synthesizing cell compartments of Saccharomyces cerevisiae. To investigate the mechanism of serylation, structural organization of the enzymes and the mode of substrate recognition, we have recently complemented our research on cytoplasmic enzyme by cloning and expression of the gene for mitochondrial seryl-tRNA synthetase. There is only 26 % similarity in the primary structure of two SerRS proteins. Determination of substrate binding domains by alignment guided mutagenesis, performed recently at cytoplasmic SerRS (Lenhard, B., Filipi}, S., Landeka, I., [krti}, I., Söll, D. and Weygand-\ura{evi}, I. (1997) J. Biol. Chem. 272, 1136-1141), has been extended to analyze the structure/function relationship in mitochondrial enzyme. This is of particular interest as the substrate specificities of these enzymes must differ sufficiently to foil confusion between organellar and cytoplasmic protein synthesis. The crystal structure of prokaryotic SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842) allowed us to produce structural models for yeast seryl-tRNA synthatases and to place the mutations in a structural context. In conjunction with structural data for Thermus thermophilus SerRS, our kinetic experiments suggest that yeast cytoplasmic enzyme displays tRNA-dependent serine recognition, affected by the mutations in motif 2 loop residues. We are trying to elucidate whether mitochondrial SerRS structurally and functionally resembles its cytoplasmic counterpart.

The formation of aminoacyl-tRNA, catalyzed by aminoacyl-tRNA synthetases, is a crucial step in maintaining the fidelity of protein biosynthesis. Sequence-specific interactions between these enzymes and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of non-cognate substrates. An investigation of the structure/function relationship in Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) revealed that RNA:protein interactions also affect the amino acid affinity of the enzyme. Yeast cytoplasmic SerRS is a homodimeric class II aminoacyl-tRNA synthetase. It shares significant sequence homology with and has the same oligomeric structure as its prokaryotic counterparts, which were crystallized in other laboratories. This facilitated yeast SerRS modeling studies and the alignment-guided mutagenesis of its SES1 gene. We examined the effects of amino acid alterations in the active site and the truncation of the C-terminal non-catalytic domain (which characterizes only eukaryotic SerRS enzymes) on the substrate recognition. Steady-state kinetic analyses of the purified mutant SerRS proteins, in conjunction with in vivo complementation studies, suggest that yeast seryl-tRNA synthetase undergoes a conformational change induced by positioning the 3'end of tRNA into the active site. Even subtle perturbation of SerRS structure interfere with the proposed structural flexibility of the enzyme, which may lower the accuracy of aminoacylation. In order to determine whether tRNA-dependent amino acid recognition is a general property of serine system, we have recently cloned and overexpressed two organellar seryl-tRNA synthetases (one from yeast and the other from maize) whose catalytic mechanisms are currently under the study.