Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa. [Display omitted] •PaIch and PaCcl are important for maintaining intracellular itaconate homeostasis•Crystal structures of PaIch and PaCcl•PaIch contains a distinct catalytic motif and substrate-binding pocket•PaCcl uses a specialized interaction network to stabilize its closed conformation Huang et al. elucidate the important roles of PaIch and PaCcl in maintaining itaconate homeostasis. The reported crystal structures reveal PaIch’s distinctive catalytic motif and substrate-binding pocket and the interaction network in PaCcl to stabilize its closed conformation, providing insights into the evolution of these enzymes to adapt to itaconate.