A Mammalian Type I Fatty Acid Synthase Acyl Carrier Protein Domain Does Not Sequester Acyl Chains

Płoskoń, Eliza; Arthur, Christopher J.; Evans, Simon; Williams, Christopher; Crosby, John; Simpson, Thomas J.; Crump, Matthew P.

doi:10.1074/jbc.m703454200

Cited by 75 publications

(110 citation statements)

References 56 publications

(64 reference statements)

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“…Although KS domains are dimeric in both Type I and Type II PKSs, in the latter the KS exists as a discrete protein without a covalently attached KS-AT linker or AT domain. This suggests that the mode of KS-ACP recognition is different in these two systems and adds to a growing body of evidence suggesting important mechanistic differences between type I and II systems (31).…”

Section: Resultsmentioning

confidence: 96%

“…In contrast the [KS3][AT3] didomain crystal structure (38) lacks the N-terminal docking domain and has an irreversible inhibitor bound at the active site of the KS domain. The apo form of the ACP was used because it has been shown that for type I systems apo-, holo-, and acyl-ACPs are conformationally similar (31,39). An additional distance constraint was applied such that the conserved serine on the ACP domain was between 12-25 Å of the active site cysteine of the KS domain to satisfy the constraint imposed by the length of the phosphopantetheine arm and search the KS surface more efficiently.…”

Section: Methodsmentioning

confidence: 99%

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Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Kapur

Chen²,

Cane³

et al. 2010

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

115

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Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.assembly line enzymes | polyketide synthase | protein-protein interactions M odular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) are assembly lines of catalytic modules responsible for the biosynthesis of polyketide natural products (1-6). A fundamental challenge toward elucidating their molecular logic is to understand the mechanism by which covalently bound biosynthetic intermediates are sequentially accessed by individual catalytic sites of the synthase. Most notably, each module of a polyketide synthase (PKS) has an acyl carrier protein (ACP) domain that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze polyketide chain elongation, and subsequently engages with the KS domain of the next module to facilitate forward chain transfer (Fig. 1). The simplest explanation for the orderly progress of a growing polyketide chain along a multimodular PKS is that each ACP is equivalently recognized by both KS partners, and that selection of the appropriate KS-ACP pair at a given point in the catalytic cycle is solely dictated by the identity of the substrate anchored on the ACP. (In the chain elongation reaction, a nucleophilic malonyl extender unit is attached to the ACP, whereas the growing polyketide chain itself is attached to the ACP via an electrophilic thioester linkage when it is ready for forward transfer.) Previous studies, however, suggest that protein-protein recognition between the KS and the ACP domains also plays an important role in both reactions (7-10). Moreover, this protein-protein recognition not only influences the specificity (k cat ∕K M ) of each reaction, but also the maximum rate constant (k cat ).In the course of our efforts to elucidate the principles that govern KS-ACP recognition during chain elongation and chain transfer, we have made two unexpected and potentially important observations. First, notwithstanding the fact that the same active sites are deployed in both reactions, the structural elements that mediate KS-ACP recognition are entir...

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Section: Resultsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Kapur

Chen²,

Cane³

et al. 2010

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

115

View full text Add to dashboard Cite

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“…The ACP domain is located downstream of the KR domain. Based on the NMR structure of the rat FAS ACP domain and the crystal structure of the human FAS ACP domain in complex with the holo ACP synthase, ACP forms a typical four-helix bundle, with the serine for cofactor attachment preceding the second helix of the bundle (Bunkoczi et al 2007 ;Ploskon et al 2008).…”

Section: Domain Composition and Reaction Cyclementioning

confidence: 99%

Structure and function of eukaryotic fatty acid synthases

Maier

Leibundgut

Boehringer

et al. 2010

Quart. Rev. Biophys.

120

127

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Abstract. In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2Á6 MDa a 6 b 6 heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficiency.

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“…The mechanism of acyl chain interaction with the ACP molecule is remarkably different in rat, which belongs to the type I fatty acid pathway. Insignificant interactions between the ACP molecule and the acyl chain are observed, suggesting that the ACP molecule does not sequester the acyl chain, and therefore, the acyl chain in type I pathway is protected in a way different from the type II pathway (18).…”

mentioning

confidence: 99%

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway

Upadhyay

Misra

Srivastava

et al. 2009

Journal of Biological Chemistry

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Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C ␣ and C ␤ of Ser-37 in tetradecanoyl-ACP. 13 C, 15 N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C 8 )-and dodecanoyl (C 12 )-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C 14 )-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK a value for the carboxylate, ϳ1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.In the malarial parasite Plasmodium falciparum, fatty acid biosynthesis occurs by a pathway distinct from the host. A number of enzymes involved in the process viz. ␤-ketoacyl acyl carrier protein (ACP) 5 synthase III, ␤-hydroxy acyl-ACP hydratase, and enoyl-ACP reductase are targets for drug design (1, 2). An indispensable component, crucial for each step of the pathway, is a small acidic protein, the ACP. ACP plays a pivotal role in a range of biochemical processes, like fatty acid biosynthesis (3), polyketide synthesis (4, 5), oligosaccharides (6), biotin, and nonribosomal peptide synthesis (7,8). Thus, the knowledge of structural features, which dictate ACP function, could offer new avenues for inhibitor design to disable several pathways of the parasite in parallel.Acyl carrier protein differs structurally in the host and the parasite. It exists as an independent protein in type II fatty acid synthesis pathway, observed in P. falciparum, Escherichia coli, spinach, and most prokaryotes. In the type II pathway, fatty acids are synthesized by multiple enzymes catalyzing different reactions. Conversely, mammalian ACP (malarial host) is an integral domain of one single multidomain, multifunctional fatty acid synthase (FAS) (type I pathway), each domain catalyzin...

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A Mammalian Type I Fatty Acid Synthase Acyl Carrier Protein Domain Does Not Sequester Acyl Chains

Cited by 75 publications

References 56 publications

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Structure and function of eukaryotic fatty acid synthases

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway

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