Molecular basis of glutamine synthetase mediated utilization of ethanolamine from Mycobacterium tuberculosis

Glutamine synthetase (GS) is a central enzyme in nitrogen metabolism. However, the molecular mechanisms whereby mycobacteria exploit the abundant polyamines and monoamine remain largely unexplored. This study demonstrates that all four Mycobacterium tuberculosis GS isoforms exhibit catalytic activity toward polyamine and monoamine substrates, with MtbGS3 showing exceptional catalytic efficiency for utilizing ethanolamine via interactions involving critical residues S54 and E190. Cryo-electron microscopy reveals the high-resolution structures of three previously uncharacterized GS isoforms in M. tuberculosis, MtbGS2, MtbGS3, and MtbGS4, revealing distinct tetrameric, hexameric, and decameric oligomerization arrangements, respectively. Quantum mechanics/molecular mechanics (QM/MM) simulations show that ethanolamine forms stable, catalytically optimal interactions with MtbGS1 and MtbGS3 at unique active sites, while MtbGS2 and MtbGS4 exhibit suboptimal binding owing to structural defects and unfavorable ATP interactions, highlighting substrate flexibility across isoforms, particularly in ethanolamine metabolism. Phylogenetic analyses identify multiple M. tuberculosis GS isoforms across distinct branches, with MtbGS4 sharing notable similarity to eukaryotic GS, a pattern consistent with Mycobacteria's adaptive evolution to diverse niches. Collectively, these biochemical and structural insights provide the molecular basis for developing precision GS-targeted therapies.