Functional mechanism and structural dynamics of Brucella ovis UDP-N-acetylglucosamine Enolpyruvyl transferase.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for bacterial peptidoglycan biosynthesis, catalyzing the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG) to form enolpyruvyl-UDP-N-acetylglucosamine (UNAGEP). This study characterizes the kinetic properties and domain dynamics of MurA from Brucella ovis. Global kinetic analysis across varying concentrations of substrates suggests that, under turnover conditions, B. ovis MurA operates via a BiBi sequential rapid ordered mechanism, with a kcat of 90 min-1, a KmUNAG of 470 μM and a KmPEP of 30 μM. Inhibition assays demonstrate that the enzyme is targeted by the antibiotic fosfomycin, and that its efficacy is enhanced in the presence of UNAG. The integration of these kinetic and inhibition findings within a structural framework suggests that, as seen in other species, B. ovis MurA may facilitate efficient catalysis without significant inter-domain opening-closing transitions. Thus, complexes with UNAG, or with its analogues, appear to favor the covalent binding of the PEP substrate and the fosfomycin inhibitor. Furthermore, variations in catalytic efficiency and fosfomycin sensitivity among different MurA enzymes expand the diversity of the family and point to species-specific features that modulate UNAG transformation rates. Collectively, these findings advance the mechanistic understanding of MurA enzymes, particularly within the previously uncharacterized Brucella genus.