Park, Wooyoung Son, Se-Young Yi, Joon-Yeop Cha, Seungwoo Moon, Hankyeol Kim, Minyoung Ji, Sangho Yu, Wookyung Sung, Changmin Cha, Sun-Shin Hahn, Ji-Sook 2025-06-26T08:30:07Z 2025-06-26T08:30:07Z 2025-06-23 2025-06 0305-1048 https://pubs.kist.re.kr/handle/201004/152693 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 AlaRSL219M. This research elucidates the amino acid discrimination mechanism in AlaRS, independent of editing domain. Remarkably, the AlaRSL219M mutation was initially identified as a causal mutation enhancing lactate tolerance in a strain developed through adaptive laboratory evolution. We showed that AlaRSL219M 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. English Oxford University Press Unlocking the serine mischarging paradox and inhibiting lactyltransferase activity of AlaRS by a single-point mutation Article 10.1093/nar/gkaf462 1 Nucleic Acids Research, v.53, no.11 Nucleic Acids Research 53 11 Y scie scopus 001503187400001 2-s2.0-105008249897 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article METHANE DOMAIN TRANSFER-RNA-SYNTHETASE AMINO-ACID