Crystallographic analyses of four distinct structures of AnmK (anhydro-N-acetylmuramic acid kinase) from Pseudomonas aeruginosa reveal its full catalytic cycle and confirm the proposed random-sequential kinetic mechanism.

Peptidoglycan is a rigid envelope surrounding the cytoplasmic membrane of most bacterial species. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics. In a collaborative effort with the group of Shahriar Mobashery and Amr M. El-Araby (Univ. Notre Dame), and Juan A. Hermoso (IQF-CSIC) we report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. A remarkable X-ray structure for dimeric AnmK sheds light on the pre-catalytic and post-catalytic ternary complexes, one in each subunit. Computational simulations in conjunction with the four high-resolution X-ray structures reveal the full catalytic cycle. Journal of Biological Chemistry (2023) (https://doi.org/10.1016/j.jbc.2023.105198)