Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites

Parris, Kevin; Lin, Laura; Tam, Amy; Mathew, Rebecca; Hixon, Jeffrey; Stahl, Mark; Fritz, Christian; Seehra, Jasbir; Somers, W.S.

doi:10.1016/s0969-2126(00)00178-7

Cited by 220 publications

(360 citation statements)

References 43 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Buetow

Smith

Dawson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

162

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Section: Resultsmentioning

confidence: 99%

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

Buetow

Smith

Dawson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

162

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“…Helix II of several ACPs was observed to be involved in an interaction with their partner enzymes in the complex structures (9,17,18,(25)(26)(27)(28). In terms of the interaction of AT with ACP, Streptomyces coelicolor MCAT was suggested to recognize the helix II of FAS ACP, based on the previous docking and mutational studies (19).…”

Section: Discussionmentioning

confidence: 98%

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Miyanaga

Iwasawa

Shinohara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein-protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK-ACP complexes. Because transient enzyme-ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK-ACP complexes, allowing the determination of the crystal structure of the VinK-VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK-VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.polyketide | acyltransferase | acyl carrier protein | protein-protein interaction | cross-linking P olyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of various polyketide natural products (1). Bacterial modular PKSs comprise several catalytic modules that are each responsible for a single round of the polyketide chain elongation reaction. Each module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain. The AT domain recognizes a specific acyl building block and catalyzes its transfer reaction onto the 4′-phosphopantetheine arm of the ACP. KS extends the polyketide chain by condensing the resulting ACP-bound building blocks with the elongated acylACPs. Although standard modular PKSs contain AT domains in their modules, some modular PKSs lack AT domains in each module and instead receive their acyl building blocks by standalone trans-acting ATs (2).The selection of the starter unit is generally governed by the substrate specificity of the AT domain in the loading module (1). In some modular PKS systems, a didomain-type loading module comprising a loading AT domain and an ACP domain selects an acyl starter building block such as an acetate unit to generate an acyl-ACP intermediate, which is transferred to the downstream extension module for polyketide chain elongation. Alternatively, in a KS Q -type loading module consisting of three domains, the AT domain selects an α-carboxyacyl substrate such as a malonyl group, and the KS Q domain subsequently catalyzes its decarboxylation to construct an acyl-ACP thioester. For polyketide chain elongation, the AT domain of the extension module generally recognizes a specific α-carboxyacyl-CoA as an extender building block (3). Malony...

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“…This loop-2 region does not have a role in the AcpS⅐ACP binary complex (10). ACP functions to sequester the growing acyl chain attached to the prosthetic group from solvent as it shuttles the intermediates between enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

133

148

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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 ...

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Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites

Cited by 220 publications

References 43 publications

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

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

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

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

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