Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa

• Published in: The Journal of biological chemistry 2023-10, Vol.299 (10), p.105198, Article 105198
• Main Authors: El-Araby, Amr M., Jiménez-Faraco, Eva, Feltzer, Rhona, Martin-Garcia, Jose M., Karri, Bhaskara Rao, Ramachandran, Balajee, Kim, Choon, Fisher, Jed F., Hermoso, Juan A., Mobashery, Shahriar
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents; antibiotic resistance; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Catalysis; crystallography; Crystallography, X-Ray; Drug Resistance, Bacterial - genetics; Enzyme Activation - genetics; enzyme mechanism; Models, Molecular; peptidoglycan recycling; phosphoryl transfer; Phosphotransferases - genetics; Phosphotransferases - metabolism; Protein Structure, Tertiary; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2023.105198

The bacterial cell envelope is the structure with which the bacterium 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 enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the 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 precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the 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 β-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.