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

被引:3
|
作者
El-Araby, Amr M. [1 ]
Jimenez-Faraco, Eva [2 ]
Feltzer, Rhona [1 ]
Martin-Garcia, Jose M. [2 ]
Karri, Bhaskara Rao [1 ]
Ramachandran, Balajee [1 ]
Kim, Choon [1 ]
Fisher, Jed F. [1 ]
Hermoso, Juan A. [2 ]
Mobashery, Shahriar [1 ]
机构
[1] Univ Notre Dame, Dept Chem & Biochem, Notre Dame, IN 46556 USA
[2] CSIC, Dept Crystallog & Struct Biol, Inst Quim Fis Blas Cabrera, Madrid, Spain
基金
瑞士国家科学基金会; 美国国家卫生研究院;
关键词
1,6-ANHYDRO-N-ACETYLMURAMIC ACID; LYTIC TRANSGLYCOSYLASES; CELL; ULTRACENTRIFUGATION; REFINEMENT; INSIGHTS;
D O I
10.1016/j.jbc.2023.105198
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 beta-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.
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页数:12
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