Defining the Active Site of Yeast Seryl-tRNA Synthetase

Abstract

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. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.The formation of aminoacyl-tRNA, catalyzed by aminoacyltRNA synthetases, is a crucial step in maintaining the fidelity of protein biosynthesis. This family of enzymes can be partitioned into two classes of 10 enzymes each, based on conserved sequences (1) and structural motifs (2). All members of class I contain a common loop with the signature sequence KMSKS (3) and a region of homology with the HIGH peptide (4) as part of a Rossmann dinucleotide binding fold of parallel ␤-sheets (5). Class II synthetases have a different topology of dinucleotide binding based on antiparallel ␤-sheets (2, 6, 7). The three common signature motifs of class II synthetases are found in this domain. Motif 1 forms part of the conserved inter-subunit interface of homodimeric (6, 8) and heteromeric (9) synthetases. Motifs 2 and 3 contain many of the active-site residues important for ATP, amino acid, and tRNA acceptor stem recognition (10 -15). The elucidation of the crucial role of sequence motifs in substrate binding have resulted from the solution of several crystal structures of enzymes and enzyme-substrate complexes from both class I and class II (16) and numerous biochemical studies involving mutant synthetases (17-22).The evolution of tRNA recognition systems has recently gained much attention (23-27). The primary structure of several prokaryotic and eukaryotic seryl-tRNA synthetases (Ref.23; see also the legends to Fig. 1 and 3), including the enzyme that probably functions in yeast mitochondria (28), have been determi...

Trypanosoma Seryl-tRNA Synthetase Is a Metazoan-like Enzyme with High Affinity for tRNASec

“…In addition to this domain, the structures of most SerRSs include a coiled-coil domain at the amino end of their sequences and, in the case of eukaryotic enzymes, a C-terminal extension that plays a modest role on protein stability and amino acid recognition (7,8). The N-terminal coiled-coil domain is essential for the recognition of the elbow and, especially, the long variable loop of tRNA Ser (9,10).…”

Seryl-tRNA synthetase is not responsible for the evolution of CUG codon reassignment inCandida albicans

“…The N‐terminal region of the C. albicans SerRS contains four putative α‐helices which are likely to be responsible for tRNA recognition (Price et al ., 1993; Biou et al ., 1994; Cusack et al ., 1996) and share 63% identity with the corresponding region in the S. cerevisiae SerRS. The N‐terminal antiparallel α‐helical domains of the SerRS are conserved throughout evolution (Figure 2; Lenhard et al ., 1997, 1999). The sequence variation which occurs within the N‐terminal domain of the S. cerevisiae and C. albicans SerRS may reflect the different selective pressures that have directed the co‐evolution of the ability of the CaSerRS to discriminate different tRNAs and tRNA identity elements.…”

“…The motif 2 loop, which lies within the class II signature sequence, is involved in the binding of the ATP, amino acid and the acceptor stem of the tRNA (Lenhard et al ., 1997). The critical residues within the motif 2 loop in S. cerevisiae (R279, E281, G291; Lenhard et al ., 1997) are conserved within the C. albicans motif 2 loop (Figure 2). The only major difference between the C. albicans and S. cerevisiae SerRS primary amino acid sequences in this region is the inclusion of I282 instead of V282 in the C. albicans sequence.…”

Discovering New Classes of Brugia malayi Asparaginyl-tRNA Synthetase Inhibitors and Relating Specificity to Conformational Change

“…This loop is involved in the binding of ATP and the acceptor end of the cognate tRNA and has been reported to play a significant role in conformational changes associated with other class II AARS, such as AspRS [74], LysRS [75], SerRS [76], ProRS [77] and HisRS [78]. Mutations in this loop have also been shown to affect the tRNA dependent amino acid recognition by SerRS [79]. Motion of this loop in Brugia AsnRS, as simulated by ROCK, exposes a new cavity near the adenosine pocket of the binding site, leading to an open conformation that emulates the apo crystal structure of the protein (Fig.…”