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LC‐MS assay targeting the mycobacterial indirect aminoacylation pathway uncovers glutaminase activities of the nondiscriminating aspartyl‐synthetase
Author(s) -
Chew Bing Liang Alvin,
Tanoto Frederick Reinhart,
Luo Dahai
Publication year - 2020
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13786
Subject(s) - aminoacylation , glutaminase , biochemistry , enzyme , chemistry , microbiology and biotechnology , biology , transfer rna , rna , glutamine , amino acid , gene
The synthesis of asparagine (Asn)‐tRNA Asn 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.