Coupling between substrate specificity and neutralisation mechanisms in toxic Small Alarmone Synthetases

Abstract Toxic Small Alarmone Synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin (TA) and secretion systems. toxSASs act either through translation inhibition mediated by pyrophosphorylation of tRNA CCA-ends (exemplified by FaRel2) or through synthesis of the toxic alarmone (pp)pApp and ATP depletion (exemplified by FaRel). Both enzymatic reactions use ATP as the pyrophosphate donor. Here we show that the pseudo-Zn2+ finger domain (pZFD, equivalent to Panacea-Associated Domain 1, PAD1) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensible for FaRel neutralisation. Collectively our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity.