Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8°C, which is much higher than that of Escherichia coli ACP (67.2°C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four ␣-helices (helix I (residues 3-17), helix II (residues 39 -53), helix III (residues 60 -64), and helix IV (residues 68 -78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe 45 in helix II and Phe 18 in the ␣ 1 ␣ 2 loop and a hydrogen bonding between Ser 15 in helix I and Ile 20 in the ␣ 1 ␣ 2 loop, resulting in its high thermal stability. Phe 45 -mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser 58 in the ␣ 2 ␣ 3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.

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