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

Abstract

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 ...

Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

“…ACP is highly acidic and requires an electropositive surface on its cognate partners for binding. Chemical modification of arginine or lysine residues on LpxA compromises the acyl transferase activity (16), and, in FA biosynthesis, interactions of ACP with FabG, FabH, and AcpS are dependent on specific arginine residues (20)(21)(22). Like other ACP-targeted enzymes, LpxD presents an electropositive surface near the active site (SI Fig.…”

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

“…These divalent cation-binding sites lie along helix II of ACP. Various binding, mutagenesis, and docking studies have implicated helix II of ACP to be the "recognition helix" that facilitates interaction with various enzymes (33)(34)(35)(36). The recognition helix contains two acidic sub-sites: Site A, composed of residues E30, D35, D38 and E41, and Site B, composed of E47, D51, E53, and D56 (51).…”

Insights into mitochondrial fatty acid synthesis from the structure of heterotetrameric 3-ketoacyl-ACP reductase/3R-hydroxyacyl-CoA dehydrogenase

“…For mtFAS, the substrates are fatty acyl conjugates of ACP, similar to that for bacterial FAS-II. ACP is highly conserved in amino acid sequence, especially, the helix a2, which is at the ACP-enzyme interaction site 30 . The identity of amino acid sequences of the helix a2 between E. coli, Saccharomyces cerevisiae (S. cerevisiae) and human is 70% (Fig.…”

“…To experimentally test these predictions, we created a number of single and multiple mutated variants of HsKAR (Supplementary Tables 3 and 4). The arginine residues Arg129 and Arg172 in E. coli fabG have been demonstrated to be involved in ACP binding only by in vitro tests 30 . Therefore, the variants fabG-R129E and fabG-R172E of E. coli fabG were re-investigated using our yeast in vivo system (Fig.…”

“…In E. coli fabG, two arginine residues, Arg129 and Arg172, were found to play an important role in ACP recognition and binding 30 . In enzyme NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5805 ARTICLE activity assays, E. coli fabG variants fabG-R129E and fabG-R172E showed 8.5% and 3.5% of ACP-dependent activity, respectively, while CoA-dependent activities were largely unaffected 30 . These arginines are well conserved in most of the fabG-type enzymes.…”