Wooldridge, R. | bioRxiv Science

bioRxiv2025

Efficient genetic code expansion tools enable in vivo study of lysine acetylation in non-model bacteria

Recent proteomic advancements have revealed widespread N{varepsilon}-lysine acetylation in pathways governing pathogenicity, metabolism, and antibiotic resistance in bacteria. The spontaneous, non-specific nature of this modification in prokaryotes obscures its biological role, necessitating prokaryotic specific in vivo interrogation systems. Genetic Code Expansion (GCE) offers a powerful method to investigate the roles and regulation dynamics of acetyl-lysine in vivo with the precise incorporation of a suite of non-canonical amino acids, including acetyl-lysine analogs. However, its use has been largely restricted to E. coli strains due to challenges associated with implementation and optimization of the technology in more diverse bacterial strains. Here, we present a bacterial host-agnostic, readily optimizable GCE platform designed to site-specifically incorporate non-canonical amino acids into target proteins within living bacteria. We further demonstrate the versatility of this technology by showcasing, for the first time, the successful incorporation of acetyl-lysine in a non-E. coli bacterium.

synthetic biology↗

bioRxiv2022

CADD from Chlamydia trachomatis is a manganese-dependent oxygenase that employs a self-sacrificing mechanism for the synthesis of p-aminobenzoate

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate synthase involved in a non-canonical route for tetrahydrofolate biosynthesis in the intracellular bacterial pathogen, Chlamydia trachomatis. The previously solved crystal structure revealed a seven-helix bundle architecture similar to heme oxygenase with a diiron active site, making CADD a founding member of the emerging HDO (heme-oxygenase-like diiron oxidase) superfamily. The CADD-dependent route for pAB biosynthesis was shown to use L-tyrosine as a precursor, however, in vitro reactions were not stimulated by the addition of free L-tyrosine or other tyrosine-derived metabolites, leading to the proposal that the enzyme uses an internal active site tyrosine residue as a precursor to pAB. Here, we perform further biochemical characterization of CADD to clarify the details of the unique self-sacrificing reaction. Surprisingly, the pAB synthase reaction was shown to be dependent on manganese as opposed to iron and the data are most consistent with an active dimanganese cofactor analogous to class Ib and class Id ribonucleotide reductases. Experiments with 18O2 demonstrated the incorporation of two oxygen atoms from molecular oxygen into the pAB product, supporting the proposed mechanism requiring two monooxygenase reactions. Mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, a modification that was increased in reactions that produced pAB in vitro. Additionally, Lys152 was found to be deaminated and oxidized to aminoadipic acid. Taken together, our results support the conclusion that CADD is a manganese-dependent oxygenase that uses Tyr27 and possibly Lys152 as sacrificial substrates for pAB biosynthesis.

biochemistry↗