Unlocking the serine mischarging paradox and inhibiting lactyltransferase activity of AlaRS by a single-point mutation

Aminoacyl-tRNA synthetases are critical for accurate genetic translation, attaching amino acids to their corresponding transfer RNA molecules. Alanyl-tRNA synthetase (AlaRS) often misactivates Ser or Gly instead of Ala, which is detrimental unless corrected by its editing functions. The paradox of misactivating larger Ser by AlaRS was considered inevitable due to its inherent design, sharing an essential acidic residue to accommodate the activated adenylated intermediates from both cognate and non-cognate amino acids. Here we show a groundbreaking discovery where a single-point mutation, L219M, in AlaRS from Methylomonas sp. DH-1, effectively eliminates Ser misactivation. Structural analysis of the pre-activation state unveiled that the flexibility of Val204 is the key to preventing Ser binding in AlaRS^L219M. This research elucidates the amino acid discrimination mechanism in AlaRS, independent of editing domain. Remarkably, the AlaRS^L219M mutation was initially identified as a causal mutation enhancing lactate tolerance in a strain developed through adaptive laboratory evolution. We showed that AlaRS^L219M also eliminates the enzyme's inherent lactyltransferase activity, suggesting that the lactate tolerance observed might result from preventing excessive protein lactylation under lactate stress. This opens possibilities for developing high-fidelity and lactylation-deficient AlaRS mutants across various organisms, facilitating studies on their potential benefits in different physiological scenarios.