LC-MS assay targeting the mycobacterial indirect aminoacylation pathway uncovers glutaminase activities of the nondiscriminating aspartyl-synthetase.

The synthesis of asparagine (Asn)-tRNAAsn in most prokaryotes uses an indirect aminoacylation pathway involving a nondiscriminating aspartyl synthetase (ND-AspRS) and a glutamine amidotransferase (GatCAB). This was recently implicated as an adaptive mistranslation mechanism for antimicrobial resistance in Mycobacterium tuberculosis, but it remains poorly understood. We report an accessible liquid chromatography-mass spectrometry method with unparalleled chemical specificity, sensitivity, and quantification over the current assays to enable the direct analysis and drug screening campaigns of this pathway. Through this method, we show that the mycobacterial ND-AspRS stimulates the glutaminase activity of GatCAB. We further uncover novel glutaminase activity of the synthetase. These biological insights help better understand the indirect aminoacylation biology and allude to new roles beyond protein translation.