Inducible auto-phosphorylation regulates a widespread family of nucleotidyltransferase toxins

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA₁ induces auto-phosphorylation of MenT₁ by repositioning the MenT₁ phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA₃ is similarly able to induce auto-phosphorylation of cognate toxin MenT₃. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.