Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation.

Peptidoglycan (PG) is the main constituent of the bacterial cell wall. The enzyme UDP‑N‑acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridinediphospho‑N‑acetylglucosamine, which is the first committed step of PG biosynthesis. In this study, we have systematically examined the urea-induced unfolding of Acinetobacter baumannii MurA (AbMurA) using various optical spectroscopic techniques and molecular dynamics (MD) simulations. The urea-induced unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated between 3.0 and 4.0 M. Above 6.0 M urea, AbMurA gets completely unfolded. The transition from the native structure to the partially unfolded metastable state involves ~30% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes AbMurA structure weakening hydrophobic interactions and the hydrogen bond network. We observed a clear correlation between both in vitro and in silico studies. To our knowledge, this is also the first report on unfolding/stability analysis of any MurA enzyme.