Dynamics of acyl carrier protein in de novo fatty acid synthesis by Enterococcus faecalis based on NMR spectroscopy and molecular dynamics simulation

Abstract Fatty acid synthesis (FAS) is essential for the production of biological components such as cell membrane building blocks and metabolism-related compounds. There are two types of bacterial FAS: de novo FAS and FAS through the incorporation of external fatty acids. Enterococcus faecalis possesses two distinct acyl carrier proteins (ACPs), AcpA (EfAcpA) and AcpB (EfAcpB), which serve as cofactors in the two types of FAS. We previously showed through NMR spectroscopy that EfAcpA comprises only three long helices, while EfAcpB consists of four helices, including a short α 3 helix, similar to other bacterial ACPs. An increase in melting temperature (T m ) from 64.0 to 76.1 °C confirmed that protein structural stability increased in the presence of calcium ions. Using NMR spectroscopy, two metal binding sites were identified in EfAcpA: site A was located at the start of the α 2 helix while site B was situated near the α 2 helix and α 2 α 3 loop. To understand the importance of structural flexibility of EfAcpA in de novo FAS, we investigated its motional properties using backbone spin relaxation and molecular dynamics simulations. The α 2 α 3 loop in EfAcpA displayed high flexibility, as indicated by low heteronuclear NOE values. The residues Val51, Glu54, and Gly58 exhibited significant R 2 values, likely due to the movement of this loop. EfAcpA created a novel cavity towards the α 1 α 2 loop, in contrast to conventional cavity formation in most bacterial ACPs. This unique behavior was attributed to the flexibility exhibited by the α 2 α 3 loop. The structural and motional characteristics of EfAcpA confirmed that its conformational plasticity is a crucial factor influencing acyl chain transfers in de novo FAS. Given the increasing antibiotic resistance observed for E. faecalis in clinical settings, the findings of this study may contribute to the development of more effective pathogen management strategies targeting FAS.