Acyl carrier protein from E. coli. Structural characterization of short-chain acylated acyl carrier proteins by NMR

Mayo, Kevin H.; Prestegard, James H.

doi:10.1021/bi00347a049

Cited by 52 publications

(72 citation statements)

References 17 publications

(33 reference statements)

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“…This result suggests that different conformational changes accompany conversion of holo-ACP to 10:0-ACP and 18:0-ACP. This idea is supported by previous work on E coli acyl-ACP with short (2-8 carbon) acyl chains, which suggested that acylation causes a conformational change in this region (15). In addition, our CastP cavity results indicate that the G53-D57 loop region undergoes a conformational change to accommodate fatty acids larger than 10 carbons.…”

Section: Discussionsupporting

confidence: 88%

“…This work follows up on the earlier observation of large differences in the 15 N HSQC spectra of 10:0-and 18:0-ACP (19) and is a step toward future investigation of ACP-Δ9D interactions.…”

mentioning

confidence: 83%

“…[U- 15 N]-labeled 10:0-and 18:0-ACP were produced as previously described (19), and NMR samples were prepared at protein concentrations of 2 mM. The same expression system, but with 99% [U- 13 C]-glucose (Cambridge Isotope Laboratories, Andover, MA) as the sole carbon source during stirred-vessel fermentation, was used to produce >98% [U- 13 C, U-15 N]-labeled ACP.…”

Section: Experimental Procedures Sample Preparationmentioning

confidence: 99%

“…Several multinuclear datasets were collected for backbone and sidechain assignment of both 10:0-and 18:0-ACP: HNCO, HNCACB, C(CO)NH, H(CCO)NH, H(C)CH TOCSY, and TOCSY- 15 The NMRPipe (22) software suite was used to process the raw datasets. Generally, each dimension was subjected to the following processing steps: linear prediction (when necessary) to estimate missing initial points, cosine window function, zero filling, Fourier transform, phase adjustment, and baseline correction (in some cases).…”

Section: Data Collection For the Nmr Structural Studiesmentioning

confidence: 99%

“…Analysis of a series of 15 N HSQC spectra of acyl-ACP collected as a function of time revealed the appearance of peaks at the positions of those of holo-ACP. The peaks that grew in had 1 H linewidths of around 15 Hz in contrast to the 18-23 Hz linewidth of the acyl-ACP peaks.…”

Section: Conformational Differences Betweenmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 88%

“…This work follows up on the earlier observation of large differences in the 15 N HSQC spectra of 10:0-and 18:0-ACP (19) and is a step toward future investigation of ACP-Δ9D interactions.…”

mentioning

confidence: 83%

Section: Experimental Procedures Sample Preparationmentioning

confidence: 99%

Section: Data Collection For the Nmr Structural Studiesmentioning

confidence: 99%

Section: Conformational Differences Betweenmentioning

confidence: 99%

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

2006

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Desaturases fused to their electron donor

Sperling¹,

Heinz²

2001

Eur. J. Lipid Sci. Technol.

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Fatty acid desaturations in the carboxy‐terminal segment from C1—C10 are catalyzed in many, but not in all cases, by desaturase enzymes which are fused to their electron donor cytochrome b5. Several of these enzymes (“front‐end desaturases”) from a wide variety of organisms have been cloned and functionally expressed for proof of regio‐, stereo‐ and chain length‐selectivity. In most cases the actual status of the substrate chain, whether coenzyme A thioester or component of a membrane lipid, is not known. The cytochrome b5 domain is located N‐terminally, internally or C‐terminally. Compared to the free cytochrome b5 , the fused domains show a significant reduction of acidic amino acid residues on the surface of the four helices enclosing the heme group. It is discussed how this may contribute to hydrophobic domain pairing required for interdomain electron transport. This is in contrast to the mode of interaction of free cytochrome b5 with its partners, which is governed by electrostatic charge pairing. A look at crystallized or computer‐simulated models involving fused or free cytochrome b5 helps to outline the problems encountered by optimizing the docking of partners and the exchange of electrons between domains of different degrees of mobility.

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Hydrodynamic Properties ofVibrio harveyiAcyl Carrier Protein and Its Fatty-Acylated Derivatives

Shen

Byers

1997

Archives of Biochemistry and Biophysics

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Acyl carrier protein from E. coli. Structural characterization of short-chain acylated acyl carrier proteins by NMR

Cited by 52 publications

References 17 publications

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

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

Desaturases fused to their electron donor

Hydrodynamic Properties ofVibrio harveyiAcyl Carrier Protein and Its Fatty-Acylated Derivatives

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Acyl carrier protein from E. coli. Structural characterization of short-chain acylated acyl carrier proteins by NMR

Cited by 52 publications

References 17 publications

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

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

Desaturases fused to their electron donor

Hydrodynamic Properties ofVibrio harveyiAcyl Carrier Protein and Its Fatty-Acylated Derivatives

Contact Info

Product

Resources

About

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

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