Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Holak, T.A.; Kearsley, Simon K.; Kim, Y.; Prestegard, James H.

doi:10.1021/bi00416a046

Cited by 108 publications

(102 citation statements)

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“…The E. coli ACP is a highly acidic protein of 77 residues. It is composed primarily of a 3-helix bundle (3,4), and recent structural studies reinforce the notion that many ACPs from different synthases and/or organisms are of similar size and structure to E. coli ACP (5-7).…”

mentioning

confidence: 67%

“…Using the entire acpS gene as the probe, the MudJ insertion into the acpS gene was confirmed. 3 Because suppressor mutants of E. coli can be isolated despite the lack of functional AcpS, alternative post-translational modification enzyme(s) for apoACP must exist in the organism.…”

Section: Discussionmentioning

confidence: 99%

“…Nonetheless, acpS suppression by derepression of entD by iron starvation was examined, and no suppression of acpS was found under these conditions. 3 Cloning of entD under an inducible promoter and testing its ability to suppress an acpS Ϫ phenotype gave a negative result, 3 despite the fact that the EntD that was expressed was capable of phosphopantetheinylating EntF in crude cell extracts. This provides further evidence that entD is not able to suppress acpS and modify apoACP in vivo.…”

Section: Discussionmentioning

confidence: 99%

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Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl Transfer in Escherichia coli

Flugel

Hwangbo

Lambalot

et al. 2000

Journal of Biological Chemistry

129

134

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Holo-(acyl carrier protein) synthase (AcpS) posttranslationally modifies apoacyl carrier protein (apoACP) via transfer of 4-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 ␥-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth. Earlier mutagenic studies isolated the E. coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis. Sequencing of the acpS gene from the E. coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a ϳ5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E. coli MP4 strain phenotype. It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E. coli MP4 strain. In addition, it is demonstrated that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E. coli.Escherichia coli utilizes a repeated cycle of condensation, reduction, dehydration, and isomerization reactions to produce saturated and unsaturated fatty acids (1, 2). This biosynthetic mechanism employs multiple enzymatic activities conglomerately termed the fatty acid synthase. The central coenzyme in all fatty acid synthases is the holo form of acyl carrier protein.ACPs, 1 as free proteins or as domains of large multifunctional proteins, function in a variety of synthases as hubs to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final compound. The apo to holo conversion of ACPs by post-translational modification with the 4Ј-phosphopantetheine (4Ј-PP) prosthetic group is essential to their function, since all acyl chain molecules undergoing biosynthesis by these systems are attached to the terminal cysteamine thiol of 4Ј-PP via a thioester bond.The E. coli fatty acid synthase has served as a model system for understanding the 4Ј-PP post-translational modification, structure, and function of ACPs. The E. coli ACP is a highly acidic protein of 77 residues. It is composed primarily of a 3-helix bundle (3, 4), and recent structural studies reinforce the notion that many ACPs from different synthases and/or organisms are of similar size and structure to E. coli ACP (5-7).E. coli ACP is post-translationally modified by holo-(acyl carrier protein) synthase (EC 2.7.8.7) in a Mg 2ϩ -dependent reaction, where the 4Ј-PP moiety is transferred from CoA to the conserved serine 36 ␥-OH of ACP (8). AcpS therefore converts inactive apoACP to its activated holo form. HoloACP is then capable of being a...

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mentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl Transfer in Escherichia coli

Flugel

Hwangbo

Lambalot

et al. 2000

Journal of Biological Chemistry

129

134

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“…It is unclear whether this inhibition reflects interactions between the apoACP protein and MAT or KS/CLF or both. Since the solution structures of several Type II ACPs have been solved (31)(32)(33)(34)(35)(36)(37)(38), further studies into the structural basis for these molecular recognition properties could provide fundamentally new insights into the importance of protein-protein interactions in regulating Type II PKS function and specificity.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Dreier¹,

Shah²,

Khosla³

1999

Journal of Biological Chemistry

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Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA: ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-␤-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of proteinprotein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 ؎ 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [ 14 C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

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“…ACP contains such a dipeptide sequence for Pro55 and Asp56. Moreover, the amino acid sequence Thr52, Glu53, Ile54, Pro55 forms a turn which is flanked on both sides by helices of residues 56 -63 and residues 37 -47 [28]. The second binding site is located in a righthanded loop domain on the N-terminal side of the a helix involving residues 37-51 [13].…”

Section: Discussionmentioning

confidence: 99%

Divalent cation biding to reduced and octanoyl acyl‐carrier protein

Tener

Mayo

1990

European Journal of Biochemistry

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Mn(I1) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average &/site z 80 pM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e. g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average &/site z 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 'H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.Fatty acid biosynthesis is a central biochemical pathway involving several enzymatic steps [I -31. Acyl-carrier proteins (ACP) are coenzymes which function in acyl-group-transfer reactions, chain-elongation reactions by successive addition of two-carbon fragments from malonyl-CoA, and as anchors for reduction and dehydration reactions that ultimately lead to a saturated fatty acid [2]. Since ACP is active in a variety of differing acylated states involved in various enzymatic reactions and, therefore, with a number of different enzymes, it is likely that ACP has the ability to modify or fine-tune its conformation to interact efficiently in all situations and, perhaps, even effect regulation of fatty acid biosynthesis.The ability to isolate ACP from Escherichiu coli as a soluble protein, and not as part of a tightly associated protein complex found in higher organisms, has made it the most studied among all ACPs which, in any event, possess a high degree of sequence similarity [2]. Of the 77 amino acids (8847 Da) found in E. coli ACP, 22 are acidic (glutamic and aspartic acid residues) and six are basic (lysine, arginine and histidine residues) [4, 51. This gives the protein an net charge of -16. To date, a secondary backbone structure for E. coli ACP-SH (reduced) has been derived from two-dimensional 'H-NMR studies [6]. The following segments are proposed to be helical: residues 3 -15,37 -51,56 -63 and 65 -75; these are connected by extended chain and p-turn structure. The three-dimensional structure of ACP-SH, determined by NMR pseudo-energy and distance geometry calculations [7], have identified three helical regions: residues 3-14, 37-51 and 65-75, which run anti-parallel to each other. Octanoyl-ACP, an S-acylated species of ACP, shows a similar overall conformation with Correspondence to K .

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Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Cited by 108 publications

References 29 publications

Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl Transfer in Escherichia coli

Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl Transfer in Escherichia coli

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Divalent cation biding to reduced and octanoyl acyl‐carrier protein

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