Résumé
SignificanceEnzyme I (EI) is a central player in bacterial metabolism that controls carbon uptake, virulence, and biofilm formation of bacterial cells. Conserved in gram-positive and gram-negative bacteria but absent in eukaryotes, EI is a promising target for antimicrobial design. EI’s monomer–dimer equilibrium plays a crucial role in regulating enzymatic activity, yet very little is known about the changes in structure, dynamics, and substrate-binding properties undergone upon dimerization. Using a combination of protein engineering and pressure perturbation, we isolated EI’s monomeric state and characterized its structure in solution by NMR spectroscopy. Our study provides an atomic-level description of the structural changes accompanying EI dimerization and opens perspectives for the design of allosteric inhibitors.
Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer–dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI’s monomeric state have yet been hampered by the dimer’s high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI’s monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.