Acyl carrier protein from Escherichia coli: characterization by proton and fluorine-19 nuclear magnetic resonance and evidence for restricted mobility of the fatty acid chain in tetradecanoyl-acyl-carrier protein

Gally, Hans Ulrich; Spencer, A. K.; Armitage, Ian M.; Prestegard, James H.; Cronan, John E.

doi:10.1021/bi00618a009

Cited by 23 publications

(7 citation statements)

References 21 publications

(22 reference statements)

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“…Primarily on the basis of hydrophobic binding studies of acyl-ACPs to octyl-Sepharose, Cronan (1983) has suggested the presence of an acyl-chain binding site able to shield from six to eight carbons of a fatty acid chain from interaction with hydrophobic chromatography resins. This hypothesis of an acyl chain binding site has also been suggested by 19F NMR studies on 19F-labeled fatty acids bound to ACP (Galley et al, 1978). The tertiary structural model of ACP-SH (Mayo et al, 1983) is consistent with the proposal of a specific hydrophobic binding site since a hydrophobic cleft formed by the arrangement of the four -helices is present in the model.…”

supporting

confidence: 79%

“…This hypothesis of an acyl chain binding site has also been suggested by I9F NMR studies on I9F-labeled fatty acids bound to ACP (Galley et al, 1978). The tertiary structural model of ACP-SH (Mayo et al, 1983) is consistent with the proposal of a specific hydrophobic binding site since a hydrophobic cleft formed by the arrangement of the four a-helices is present in the model.…”

supporting

confidence: 55%

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

Mayo¹,

Prestegard²

1985

Biochemistry

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Acylated acyl carrier proteins (ACPs) with acyl chain lengths of 2, 4, 6, 8, and 10 carbons were investigated by NMR and nuclear Overhauser methods at 500 MHz. Chemical shift changes of downfield aromatic and upfield, ring-current-shifted, isoleucine proton resonances monotonically vary as a function of acyl chain length with the most prominent shifts occurring with chain lengths between four and six carbons. Chemical shifts are largest for one of the two phenylalanines; however, substantial shifts do exist for Tyr-71, His-75, and two isoleucines. Since these residues are distributed throughout the molecule, their associated resonance chemical shifts are most probably explained by an induced conformational change. Comparative NOE measurements on reduced ACP (ACP-SH) and ACP-S-C8 suggest, however, that these induced conformational changes are small except for around one of the phenylalanines. A tertiary structural model for acyl-ACP consistent with our previous model for ACP-SH [Mayo, K. H., Tyrell, P. M., & Prestegard, J. H. (1983) Biochemistry 22, 4485-4493] is presented.

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supporting

confidence: 79%

supporting

confidence: 55%

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

Mayo¹,

Prestegard²

1985

Biochemistry

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“…Since the assignments of the Tyr-71 2,6 and 3,5 doublets, peaks G and I, respectively, have been made and the reso-nances are well resolved (Galley et al, 1978), these provide a logical starting point for NOE analysis. Both of these doublets were irradiated (Figure 4a,b).…”

Section: Resultsmentioning

confidence: 99%

Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

Mayo¹,

Tyrell²,

Prestegard³

1983

Biochemistry

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The downfield aromatic (6-8 ppm) and upfield ring current shifted methyl regions (1-0 ppm) in the proton nuclear magnetic resonance spectrum of acyl carrier protein (ACP) from Escherichia coli have been examined at 500 MHz by using nuclear Overhauser methods. The data are analyzed in terms of the secondary structural model of Rock & Cronan (1979) [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785], which suggests the existence of four alpha-helical segments joined by three beta-turns, and a short coil at the C terminus of the protein. Nuclear Overhauser effects among Tyr-71, Ile-69, Ile-72, and His-75 allow refinement of the secondary structure of the C terminus. Nuclear Overhauser effects among Tyr-71, Phe-28, and three Ile's also place stringent limitations on the folding of the four alpha-helices. These data allow the proposal of a tertiary structural model for ACP.

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“…It is also anticipated that conformational changes facilitate the entry and exit of the extended prosthetic group from the active site. Biophysical (18,19) and structural studies (20) point to an interaction between the acyl chain and the ACP protein. However, crystal structures reveal that the active sites of the fatty acid biosynthetic enzymes are generally located at the bottom of hydrophobic clefts or tunnels with varying dimensions (for two examples, see Refs.…”

mentioning

confidence: 99%

Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III

Zhang¹,

Rao

Heath³

et al. 2001

Journal of Biological Chemistry

167

207

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The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP⅐protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of ␤-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH⅐ACP interaction. Our experiments reveal the significance of the positively charged/ hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.The 4Ј-phosphopantetheine prosthetic group is a central and universal feature in the mechanism of fatty acid biosynthesis that provides two crucial functionalities to the process: a long and flexible arm that can reach into active sites and a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. Two types of fatty acid synthase architectures exist in nature, and the 4Ј-phosphopantetheine moiety operates quite differently in each type. The type I, or associated system, found in metazoans, consists of a single large polypeptide containing multiple active centers. In this system, the prosthetic group with its attached nascent fatty acid swings between the active sites in the multifunctional complex. This contrasts with the type II or dissociated system found in bacteria and plants in which the active centers reside in discrete protein molecules. Here, the 4Ј-phosphopantetheine moiety is covalently attached to acyl carrier protein (ACP), 1 a small protein that sequentially delivers the lipid intermediates to the active site of each enzyme in the pathway.ACP is a small, acidic and highly conserved protein with a molecular mass of 8847 Da (1). In Escherichia coli, it is encoded by the acpP gene that is located within a cluster of other fatty acid biosynthetic genes (2, 3). Biophysical (4) and solution NMR structural studies (5) show that the protein is an asymmetric monomer composed of three ␣-helices packed into a bundle with an extended and flexible loop at one end (6, 7). The fatty acid intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group, which in turn, is covalently attached to serine 36 via a phosphodiester linkage (8). In addition to fatty acid biosynthesis, ACP also supplies acyl groups for the synthesis ...

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Acyl carrier protein from Escherichia coli: characterization by proton and fluorine-19 nuclear magnetic resonance and evidence for restricted mobility of the fatty acid chain in tetradecanoyl-acyl-carrier protein

Cited by 23 publications

References 21 publications

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

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

Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III

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