Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

Abstract

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.

Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics

“…Divalent cation‐induced folding raised the melting temperatures; around 70.0 °C was reported for E. coli ACP, which is close to the T m we determined for LipD in the presence of either CaCl 2 or MgCl 2 . Higher thermal stability was also observed in ACPs from Enterococcus faecalis and Staphylococcus aureus in the presence of CaCl 2 . The increase in the melting temperature of LipD in the presence of divalent cations is also supported by our hydrogen deuterium exchange data.…”

“…Higher thermal stability was also observed in ACPs from Enterococcus faecalis and Staphylococcus aureus in the presence of CaCl 2 . 42 The increase in the melting temperature of LipD in the presence of divalent cations is also supported by our hydrogen deuterium exchange data. We observed a significant increase in the protection factors with both forms of LipD in the presence of Mg 21 (Supporting Information, Fig.…”

Structural Characterization of an ACP from Thermotoga maritima: Insights into Hyperthermal Adaptation

“…After cooling down the denatured sample to 25 • C, the α-helical structure was completely recovered, implying that the folding reaction of Tm-ACP is reversible. The specific T m of Tm-ACP was measured as 101.4 • C by DSC ( Figure 1c), which is the highest melting temperature for any ACP reported to date [15,18,34]. The reversibility was further confirmed by NMR spectroscopy, in which the 1 H-15 N heteronuclear single-quantum coherence (HSQC) spectrum of the Tm-ACP sample heated in boiling water for 15 min was identical to that of the native Tm-ACP ( Figure S1a).…”

“…The overall completeness of assignment was 93% and the quality factor (Q) of residual dipolar coupling (RDC) data was calculated as 39.52% using Q = rms(RDC measured -RDC calculated )/rms(RDC measured ) [36]. Tm-ACP consists of four α-helices (helix I (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), helix II (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54), helix III (60-65), helix IV (69-80)) connected by a long α 1 α 2 loop and two shorter loops. To accommodate the acyl chains, the hydrophobic cavities of ACPs can be expanded by protruding helix III outward [1,28,37].…”

“…A phosphopantetheine group is attached to the Ser residue (purple box in Figure 1a) by a phosphodiester linkage, and the free thiol group at the other end of the phosphopantetheine group can form a thioester bond with acyl groups [11][12][13][14]. The key structural features of various type II ACPs have been reported [15][16][17][18][19][20][21][22][23]. ACPs consist of four helical bundles connected by three loop regions, forming hydrophobic cavities to accommodate the growing acyl chains.…”