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Publication Abstract from PubMed

The bacterial cell envelope is the structure with which the bacterium both engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics, and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an important enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the remarkable pairing of hydrolytic ring-opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM and AnmK:ATP:anhNAM) 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. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. 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. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the beta-lactam imipenem compared to the wild-type strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Catalytic process of anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa.,El-Araby AM, Jimenez-Faraco E, Feltzer R, Martin-Garcia JM, Karri BR, Ramachandran B, Kim C, Fisher JF, Hermoso JA, Mobashery S J Biol Chem. 2023 Sep 1:105198. doi: 10.1016/j.jbc.2023.105198. PMID:37660917^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.