N-terminal lysine as a critical determinant of odilorhabdin susceptibility to inactivation by the self-resistance acetyltransferase OatA

ABSTRACT Odilorhabdins (ODLs) are a novel class of peptide antibiotics that inhibit bacterial translation by binding to the 30S ribosomal subunit. Xenorhabdus nematophila , an ODL-producing bacterium, encodes a highly specialized self-resistance acetyltransferase, OatA, that inactivates ODLs via primary amine modification. In this study, we investigated the structural determinants governing susceptibility of the natural compound NOSO-95C to OatA-mediated inactivation. Using an alanine-scanning library, we identified N -terminal lysine (Lys 1 ) as the key structural determinant of acetyltransferase susceptibility. Expression of oatA in E. coli increased the MIC of NOSO-95C by 64-fold due to rapid OatA-mediated acetylation. In contrast, analog 1 (Lys1→Ala) was no longer discriminated between Oat-A expressing and non-expressing strains, and remained 95.4% unacetylated after 4 h of incubation. Functional assays confirmed that analog 1 retains potent translation inhibition and antibacterial activity despite enzyme exposure. Furthermore, OatA failed to inactivate other amine-containing clinical antibiotics such as aminoglycosides, penicillin, or polymyxins. These results indicate that the side chain of N -terminal lysine is essential for efficient OatA-mediated acetylation of ODLs. Moreover, this single residue can be modified to enable ODLs to evade enzymatic inactivation while preserving their primary mechanism of action. This work provides a clear structure-activity framework for the rational design of next-generation ODLs with enhanced stability against OatA-mediated enzymatic modification.