Reprogramming a Protein Ligase for Genetic Code Expansion
Abstract
The ribosome's DNA-encoded production of defined polymer sequences is naturally limited to 22 amino acids. Although the translation machinery has the latent capacity to polymerize backbone-modified substrates, including {beta}-amino acids, this potential is constrained by the intrinsic -selectivity of native aminoacyl-tRNA synthetases. Here, we address this limitation by "reverse engineering" the Escherichia coli protein ligase EpmA. Naturally activating (R)-{beta}-lysine, EpmA evolved to discard its tRNA-binding domain in favor of protein recognition. By grafting the anticodon-binding domain of the canonical lysyl-tRNA synthetase, LysRS, onto EpmA, we created the chimeric enzyme chEpmA. To our knowledge, this represents the first successful reprogramming of a protein ligase into a functional aminoacyl-tRNA synthetase. We demonstrate that chEpmA serves as a versatile dual-specificity platform: it efficiently charges tRNAs with the non-canonical backbone (R)-{beta}-lysine, and a single substitution unlocks the scaffold for -substrates, thereby enabling a broad spectrum of post-translational modifications previously inaccessible to genetic code expansion. This repertoire ranges from acylated lysines such as N{varepsilon}-succinyl-(S)- lysine (Ksucc) and bulky modifications such as biocytin to advanced glycation end products (AGEs) including N{varepsilon}-carboxymethyl-(S)- lysine (CML). Our work establishes a structural blueprint for mobilizing non-canonical substrates, paving the way for the biosynthesis of protease-resistant peptidomimetics and next-generation therapeutics.
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