Amino Acid Residues of Escherichia coli Acyl Carrier Protein Involved in Heterologous Protein Interactions

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.

“…It has been reported that the Glu and Asp residues in helix II of Ec-ACP are important to recognize FabA, FabB, FabD, FabG, MCAT, AcpS, and FabH (61,(65)(66)(67)(68) Fig. 4A.…”

Section: Discussionmentioning

confidence: 99%

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

Park

Jung

Song

et al. 2016

“…7) suggests that FabG interactions may alter an important component of the acyl chain binding site in ACP to facilitate the release of the prosthetic group for insertion into the active site. The importance of Ile-54 is underscored by the observations that the E. coli ACP[I54C] mutant is partially defective in the reconstitution of type II fattyacid synthase in crude extracts (14), and the Vibrio harveyi ACP[I54A] mutant does not fold properly (13). Clearly, high resolution structures of various acyl-ACPs will be required to test the hypothesis that the acyl chain interacts with loop-2 (Ile-54 in particular) and whether interactions between the type II Fab proteins and loop-2 of ACP promote the release of the acyl moieties from this binding pocket.…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic analysis and direct binding studies between FabH mutants and ACP confirmed the importance of the positively charged residues to the FabH-ACP interaction. Several ACP mutants have been made, and their ability to function in type II fatty acid synthesis has been assessed (13)(14)(15). These data are consistent with the conclusion that residues along ACP helix ␣2, such as Glu-41 and Glu-48, are important for ACP function, although the binding of ACP to individual components in the crude extracts used in this work was not addressed.…”

mentioning

confidence: 99%

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

133

148

We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on ␤-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix ␣2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix ␣2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.Fatty acid biosynthesis in bacteria and plants is carried out by a series of enzymes that are collectively known as the type II fatty-acid synthase and is most extensively studied in the Escherichia coli system (1, 2). ACP 1 is a small, acidic protein that functions as the central acyl group carrier in type II fatty-acid synthase systems. The ACPs are rod-shaped proteins with a preponderance of acidic residues organized into four ␣-helices (3-7). The acyl intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group (8), which is attached via a phosphodiester linkage to Ser-36 located at the beginning of the second helical segment. The primary gene product is an apoprotein that is converted to ACP by the transfer of the 4Ј-phosphopantetheine moiety of CoA to Ser-36 by [ACP]synthase (AcpS) (9, 10). ACP performs two functions. First, it sequesters the growing acyl chain from the aqueous environment, and second, upon binding to one of the type II proteins, it releases its grip on the fatty acid, which is inserted into the active site cavity of the enzyme. ACPs are widely distributed in nature and are easily recognized by their significant primary sequence identity, particularly at the prosthetic group attachment site and extending along helix ␣2 (11). These similarities are thought to underlie the observation that ACPs from virtually any source are substrates for AcpS and function with the fatty acid biosynthetic enzymes of E. coli (11). However, the individual enzymes of type II fatty acid synthesis do not share a primary structure motif that would define the presence of a common ACP-binding motif.The molecular details that govern the specific interactions between ACP and the type II enzymes are poorly known. The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant ...

“…Plasmids derived from pMR19 (10) that encode ACPs with cysteine substitutions were obtained from M. Lou Ernst-Fonberg (25). All other plasmids used in this study are listed in H]alanine (specific activity, 60 Ci/mmol; American Radiolabeled Chemicals) was used at a final concentration of 0.5 mM.…”

Section: Methodsmentioning

confidence: 99%

“…Although this system has the advantage of involving an appreciable number of enzymes, some enzymes are not required for function of the in vitro system, and because the system functions at only a few percent of the rate of fatty acid biosynthesis in vivo (17,24,25), the assay is rather insensitive and probably does not reflect rate-limiting reactions in vivo. Other studies have assayed the activity of several mutant ACPs as the substrate for a single fatty acid biosynthetic enzyme (25)(26)(27). Although these studies have provided useful data, they are of a piecemeal nature and hence often difficult to interpret in physiological terms.…”

mentioning

confidence: 99%

In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis

Lay

Cronan

2007

Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.Both type I and type II fatty acid biosynthetic systems have a key component, the acyl carrier protein (ACP), 2 that is essential for function (1-3). In both synthetic systems the ACP component carries fatty acyl intermediates among the different enzyme active sites. To perform this function ACP must be posttranslationally modified via the transfer of a 4Ј-phosphopantetheine moiety from CoA to a conserved serine residue of ACP. Attachment of this prosthetic group is mandatory because the thiol group of the phosphopantetheinyl moiety carries the intermediates of fatty acid biosynthesis. In the type I systems found in the mammalian cytosol, ACP is a domain of a very large polypeptide that contains all the enzymatic domains required for fatty acid biosynthesis (4). The functional mammalian fatty-acid synthase is a dimer of two identical polypeptides in head-to-head orientation that synthesizes the fatty acids used for complex lipid biosynthesis (5-7). The finished f...