The peptidoglycan biosynthetic pathway involves a series of enzymatic reactions in which UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) plays a crucial role in catalyzing the conversion of UDP-N-acetylglucosamine-enolpyruvate (UNAGEP) to UDP-N-acetylmuramic acid. This reaction relies on NADPH and FAD and, since MurB is not found in eukaryotes, it is an attractive target for the development of antimicrobials. MurB from Brucella ovis, the causative agent of brucellosis in sheep, is characterized here. The FAD cofactor in MurB of B. ovis is reduced to the hydroquinone state without semiquinone stabilization with an estimated E