The N-Terminal Extension of the Mycobacterium avium Rel Protein Is a Dual Regulator of the Bifunctional Enzyme and Represents a Novel Target

<b>Background</b>: <i>Mycobacterium avium</i> (<i>Mav</i>) is a leading cause of pulmonary disease among non-tuberculous mycobacteria (NTMs) due to its extensive antibiotic resistance profile. The essential Rel protein is a bifunctional enzyme, which is sensitive to environmental stress and regulates cellular guanosine-3',5'-bispyrophosphate ((p)ppGpp). Increased levels of the alarmone thereby initiate a survival response, contributing to bacterial persistence and virulence. <b>Objectives</b>: <i>Mav</i>Rel harbors an unusual extension at the N-terminal domain (NTD), which we aim to characterize its possible regulatory role in maintaining (p)ppGpp homeostasis. We also studied whether the TGS domain retains its regulation capacity in <i>Mav</i>Rel and the binding propensity of the ACT domain to valine. <b>Methods</b>: Molecular dissection of <i>Mav</i>Rel was performed to generate a series of truncates to quantify the synthetase and hydrolase activities. Binding experiments with tRNA and valine were carried out via tryptophan quenching assay and NMR, respectively. <b>Results</b>: Bi-catalytic regulation of <i>Mav</i>Rel was found to be predominantly governed by the residues 37-50 at the NTD extension in its free state. The TGS domain was shown to harbor the capacity to bind with deacylated tRNA and represses synthetase activity to a lower degree compared to the NTD extension. We also characterized the dimeric <i>Mav</i> ACT-domain and the interacting residues contributing to its affinity with valine to function as a nutrient sensor. <b>Conclusions</b>: The mapping of the unique NTD regulatory element of <i>Mav</i>Rel reveals its functional relevance to coordinate the catalytic states of synthetase and hydrolase, hence underscores the prospect to drive inhibitor development targeting this novel site against <i>Mav</i> infections.