X-Ray Crystallographic Studies on Butyryl-ACP Reveal Flexibility of the Structure around a Putative Acyl Chain Binding Site

Roujeinikova, Anna; Baldock, Clair; Simon, William J.; Gilroy, John; Baker, Patrick J.; Stuitje, Antoine R.; Rice, David W.; Slabas, Antoni R.; Rafferty, John B.

doi:10.1016/s0969-2126(02)00775-x

Cited by 122 publications

(177 citation statements)

References 45 publications

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“…We hypothesize that this is a consequence of dynamic disorder of the phosphopantetheine in solution as observed in previous structures of ACP (41,46,17). One argument in support of phosphopantetheine dynamics involves the 1 H linewidths.…”

Section: Conformation Of the 4′-phosphopantetheinesupporting

confidence: 68%

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Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

2006

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Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo-and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0-and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0-and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners.Acyl carrier proteins participate in a wide variety of biosynthetic processes, including the synthesis of fatty acids (1), toxins (2), oligosaccharides (3,4), polyketides (5,6), biotin (7), and depsipeptides (8). They ferry small molecules between enzymes involved in biosynthesis by covalent linkage as a thioester to the thiol group of 4′-phosphopantetheine ( Figure 1). This prosthetic group is attached by a post-translational modification to a serine (S38 in spinach ACP 1 ) in the middle a conserved Asp-Ser-Leu (DSL) sequence. Whereas the sequences of different acyl carrier proteins are divergent, they share a similar four-helix bundle topology and often can be interchanged with full activity in reactions in vitro. This prompts the question: are ACPs simply passive molecules that constantly present their cargo to enzymes, or do protein-protein and protein-ligand interactions affect the utility of ACP as an enzyme substrate?One well-studied biosynthetic process involving ACP is the desaturation of the fatty acid stearate by the enzyme stearoyl-ACP Δ 9 -desaturase (Δ9D). This enzyme, in a reaction dependent on O 2 and reducing-equivalent, acts on ACP with an attached stearate (18:0-ACP) † This work was supported by the National Institutes of Health Grants R01 GM-50853 to B.G.F. and R01 GM-58667 to J.L.M. NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM), which is supported by grants from the NIH Center for Research Resources Biomedical Research Technology Program (P41 RR-02301) and the National Institutes of General Medical Sciences (P41 GM-GM66326) with additional instrumentation purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program, the NIH Shared Instrumentat...

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Section: Conformation Of the 4′-phosphopantetheinesupporting

confidence: 68%

“…Previous NMR studies of E. coli acyl-ACP (15) were of samples with shorter fatty acid chains (6-8 carbons), and the sole X-ray structure of an acyl-ACP is of E. coli butyryl-ACP (17). Spinach ACP, which has a 39% sequence identity to E. coli ACP, exhibits a similar four-helix bundle topology.…”

Section: Comparison With Other Acp Structuresmentioning

confidence: 99%

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

2006

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“…harveyi ACP° [23].°This°stabilization°occurs°as°the acyl chain is enclosed within the relatively hydrophobic interior of the three parallel ␣-helices of folded ACP, as indicated°by°both°NMR° [21]°and°X-ray°crystallography [20].°As°shown°in°Figure°5a,°the°CSD°of°myristoyl-ACP was°very°similar°to°that°of°apo-rACP°(Figure°2a)°at neutral pH (average net charge 7.6), although only the former would actually be in a folded helical conformation°in°solution°under°these°conditions° (Figure°1d).…”

Section: Resultsmentioning

confidence: 92%

“…The solvent-accessible surface area°of°E. coli butyryl-ACP°(1L0I,° [20]°was°calculated using°UCSF°Chimera°software° [27]°with°a°probe°radius of 1.4 Å.…”

Section: Methodsmentioning

confidence: 99%

“…The NMR solution structure of several ACPs reveal a highly conserved folded conformation consisting of three parallel ␣-helices; in acyl-ACP, these helices enclose a fatty acyl group that is covalently attached to the 4=-phosphopantetheine prosthetic group [20,21]. Based on circular dichroism (CD) and hydrodynamic approaches, ACP from some bacterial species such as Vibrio harveyi is largely unfolded in solution at neutral pH, and its folded conformation can be stabilized by either fatty acylation or divalent cation binding to the acidic Helix II [22][23][24].…”

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confidence: 99%

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Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Murphy

Rowland

Byers

2007

J. Am. Soc. Mass Spectrom.

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Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6 ϩ -9 ϩ species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process. (J Am Soc Mass Spectrom 2007, 18, 1525-1532

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Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

Buyachuihan,

Stegemann,

Grininger

2023

Angewandte Chemie

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Megasynthases, such as type I fatty acid and polyketide synthases (FASs and PKSs), are multienzyme complexes responsible for producing primary metabolites and complex natural products. Fatty acids (FAs) and polyketides (PKs) are built by assembling and modifying small acyl moieties in a stepwise manner. A central aspect of FA and PK biosynthesis involves the shuttling of substrates between the domains of the multienzyme complex. This essential process is mediated by small acyl carrier proteins (ACPs). The ACPs must navigate to the different catalytic domains within the multienzyme complex in a particular order to guarantee the fidelity of the biosynthesis pathway. However, the precise mechanisms underlying ACP‐mediated substrate shuttling, particularly the factors contributing to the programming of the ACP movement, still need to be fully understood. This review illustrates the current understanding of substrate shuttling, including concepts of conformational and specificity control, and proposes a confined ACP movement within type I megasynthases.

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X-Ray Crystallographic Studies on Butyryl-ACP Reveal Flexibility of the Structure around a Putative Acyl Chain Binding Site

Cited by 122 publications

References 45 publications

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

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X-Ray Crystallographic Studies on Butyryl-ACP Reveal Flexibility of the Structure around a Putative Acyl Chain Binding Site

Cited by 122 publications

References 45 publications

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate,

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate,

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

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Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,