Discrimination of Cognate and Noncognate Substrates at the Active Site of Class I Lysyl-tRNA Synthetase

Abstract

The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) the only enzyme represented in both classes. Based on the structure of L-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1), and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (k cat /K M ) with lysine by LysRS1 variants fell by 1-4 orders of magnitude compared to wild-type. Disruption of putative hydrogen-bonding interactions through replacement of G29, T31 and Y269 caused up to 1500-fold reductions in k cat /K M , similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40,000-fold reductions in k cat /K M observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than wild-type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart.Aminoacyl-tRNAs (aa-tRNAs) are synthesized when an amino acid is esterified to the 3′-end of a transfer RNA (tRNA) (1;2). After their synthesis, aa-tRNAs are screened for their correct pairing by elongation factor-TU (3) and taken to the ribosome where they base pair with the complementary mRNA and participate in protein synthesis. Correctly aminoacylated tRNAs are essential for the faithful translation of mRNA into the encoded polypeptide sequence. The aa-tRNAs are synthesized primarily by the aminoacyl-tRNA synthetases (aaRS) (4). The accuracy of aa-tRNA synthesis is ensured by the extremely high substrate specificity of the aaRSs, and this is further enhanced in some synthetases by the existence of editing mechanisms directed against noncognate substrates (5). The 20 aaRS proteins, as for example found in Escherichia coli, are divided into two mutually exclusive structural groups, comprised of ten members each, termed class I and class II (6-8). The assignment of an aaRS specific for a particular amino acid to one or the other structural class is almost completely conserved. The † This work was supported by Grant GM 65183 from the National Institutes of Health only widespread exception to this rule is the lysyl-tRNA synthetases (LysRSs). These are class I enzymes (LysRS1) in certain bacteria and archaea but are otherwise members of class II (Ly...

Small Molecules: Big Players in the Evolution of Protein Synthesis

“…The fidelity of aa recognition is dependent on the plasticity of the active site of each aaRS to accommodate the side chains without steric clashes between the substrate and the active site residues of the synthetase . Generally, each aaRS has evolved to achieve a useful level of specificity without necessarily maximizing discrimination between the cognate aa and cognate analogues . This was strikingly illustrated in a recent study that detected substrate analogues for 17 aaRSs that could be aminoacylated to cognate tRNA by the wild-type aaRSs .…”

“…AEC is potentially more problematic since, once activated by LysRS2, it can be transferred to tRNA Lys and used in protein synthesis by the ribosome, inhibiting cell (27,28). TyrRS, ThrRS, IleRS, MetRS, and TrpRS can also be found in certain genomes as apparent duplicates, and the expression of the second copy is often dependent upon the presence of inhibitors for the housekeeping version or changes in cellular physiology (45,(120)(121)(122)(123)(124).…”

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

“…While T box control of LysRS1 expression supports growth of B. subtilis , the level of charged tRNA Lys is reduced and there is a growth phenotype. However it is unclear whether this phenotype is caused by T box regulation of LysRS expression or is due to the B. cereus derived class I LysRS1 enzyme that is reported to be less efficient catalytically than its class II counterpart [ 21 ]. To distinguish between these possibilities and to further address the issue of T box regulation of LysRS, we constructed strain NF113 ( lysS ::P lysK (T box) lysS ) that placed expression of the endogenous B. subtilis lysS gene under the control of the lysK promoter and T box element from B. cereus strain 14579.…”

Class I and class II lysyl‐tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.

“…The phenotypes of the LysRS1 and LysRS2 deletion mutants are also consistent with cellular roles primarily concerned with differences in how the lysyl‐tRNA synthetases function. LysRS1 is more discriminating in its substrate specificity than LysRS2, and this trait could allow the retention of lysK in genomes (Jester et al ., 2003; Levengood et al ., 2004; Wang et al ., 2006). If this underlies the retention of lysK along with lysS in Methanosarcina spp., our current results would indicate that such misacylation is not a serious detriment to the cell under any of the growth conditions tested.…”