Pyridoxal-phosphate-dependent biosynthesis of aminovaleramide by AvaS in tRNA

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNAIle2) modified with lysidine (k2C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava2C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava2C was unknown. Here, we report that tRNAIle-aminovaleramididine synthetase (AvaS) catalyzes ava2C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k2C to ava2C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava2C-modified tRNA exhibits lower AUA decoding efficiency than k2C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava2C levels and metabolic and stress response pathways influencing i6A/ms2i6A dynamics. Together, these findings define the molecular basis of ava2C biosynthesis and its broader cellular metabolic networks.