Yeast fatty acid synthase: structure to function relationship

Singh, Nirpinder; Wakil, Salih J.; Stoops, James K

doi:10.1021/bi00344a044

Cited by 44 publications

(39 citation statements)

References 13 publications

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“…Because the active sites are completely independent functionally (35), the biochemical data supported the fidelity of the symmetric model discussed above. At the same time, however, the data did not exclude the possibility of an asymmetric molecule, especially when we consider that FAS might exist in several thermodynamically stable states.…”

Section: Discussionsupporting

confidence: 57%

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Brink

Ludtke

Yang

et al. 2001

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

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We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites. O besity, a major health factor, is a measure of the fat deposited with adipose in consequence to food intake, fatty acid and triglyceride synthesis and oxidation, and energy homeostasis (1). Excess food provides not only the energy needs of the body but promotes the synthesis of fatty acids and triglycerides and storage of the latter in liver and adipose tissues. The de novo synthesis of fatty acids from acetyl-CoA and malonylCoA in the presence of the reducing substrate NADPH is catalyzed by the enzyme fatty acid synthase (FAS, ref. 2). Malonyl-CoA is the donor of the C 2 units required for chain elongation and is generated by acetyl-CoA carboxylase reactions, the rate-limiting step in the synthesis of fatty acids. In animal tissues including that of humans, FAS is a homodimer of a multifunctional protein (M r 272,000; refs. 2 and 3). Each subunit protein contains seven catalytic activities plus the acyl carrier protein (ACP). The organization of the catalytic activities along the polypeptide chain from the N to the C termini is as follows: ␤-ketoacyl synthase, acetyl͞malonyl transacylases, ␤-hydroxyacyl dehydratase, enoyl reductase, ␤-ketoacyl reductase, ACP, and thioesterase. Throughout the elongation process, the fatty acyl groups are linked as a thioester to the 4Ј-phosphopantetheinyl-SH, the prosthetic group of the ACP component of FAS. This prosthetic group is Ϸ20 Å long and is an essential flexible linker for the catalytic activities of FAS (3, 4). The organization of the partial activities, as mentioned above, is based on proteolytic mapping of the subunit and is confirmed by the predicted amino acid sequences based on the cloning and nucleotide sequences of the cDNA of the FAS component enzymes (2, 5-11). Moreover, the multifunctional human FAS and its subdomains were cloned and expressed in Escherichia coli; the recombinant proteins were catalytically active, and their orders on the FAS subunit were established (12, 13). Based on these studies, the component activities were grouped into three functional subdomains: domain I, containing ␤-ketoacyl synthase, acetyl͞malonyl transacylases, and ␤-hydroxyacyl dehydratase; domain II, containing enoyl reductase, ␤-ketoacyl reductase, and ACP; and domain III, containing thioesterase. Moreover, an intermediate polypeptide containing 640 amino acids, located between domains I and II and designated as the int...

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Section: Discussionsupporting

confidence: 57%

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Brink

Ludtke

Yang

et al. 2001

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

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“…However, the importance of double bonds in the cerulenin side chain is also proved for E. coli enzyme [16]. The main products of yeast FAS used in this experiment are palmitoyl-CoA and stearyl-CoA under the standard turnover conditions [33], although the formation of saturated fatty-acyl groups up to C24 has been reported under nonturnover conditions usingp-nitrophenyl thioacetate as an artificial substrate [34]. In the case of stearyl-CoA biosynthesis, intermediates which bind to the cysteine thiol of the condensing enzyme have c2-c16 acyl groups (Fig.…”

Section: Discussionmentioning

confidence: 78%

Effect of side‐chain structure on inhibition of yeast fatty‐acid synthase by cerulenin analogues

Morisaki

Funabashi

Shimazawa

et al. 1993

European Journal of Biochemistry

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Yeast fatty-acid synthase (FAS) inhibition by cerulenin analogs with varying side-chain lengths was compared with that of cerulenin, tetrahydrocerulenin and iodoacetamide. Although inhibition by cerulenin was the highest, the analogs having (E,E)-A'*" double bonds showed high inhibition. This strongly suggests that the (E, E)-d7? l o double bonds play an important role in the interaction of the inhibitors with the enzyme. It was suggested that the size of the hydrophobic cavity in the condensing enzyme terminates fatty-acid chain elongation by decreasing inhibition by the C1 analog. Like cerulenin itself, the shortest analog (C,) did not induce malonyl-CoA decarboxylase activity.Cerulenin 1 [I, 21 is an antibiotic produced by Cephalosporium caerulens [3]. It has a hydrophilic head part (C1 -C4) with a cis-epoxide moiety and a hydrophobic side chain with an (E,E)-lP-diene system (C5-Cl2) [4, 51 (Fig. 1).Cerulenin inhibits fatty-acid synthase (FAS) [6] of both multifunctional and unassociated enzymes [7 -111, except the synthase from cerulenin-producing fungi [12-141. Vance et al.[15] first reported, using the enzyme preparation from Mycobacterium phlei, that 3-oxoacyl synthase (condensing enzyme) was hghly sensitive to cerulenin. Subsequently, d'Agnelo et al. [16] confirmed, with the purified enzyme from Escherichia coli, that cerulenin binds to the cysteine at the active site of the condensing enzyme (active cysteine thiol group) and inhibits the condensation of fatty-acylated acylcarrier protein (ACP) and malonyl-ACP. They observed that inhibition of the condensing enzyme by cerulenin was irreversible, and that the molar ratio of cerulenin/enzyme was approximately 1 : 1 when inhibition approached 100%.Inhibition of FAS by cerulenin is more potent and specific [7] than that by the thiol inhibitor iodoacetamide (4), which binds to the same cysteine residue [17-191. This was demonstrated by comparison of the second-order rate constants (k,) of the reactions. The values of the rate constant k, were 88 M-' . S-' [7] and 1.0 M-' . S- ' [19] for the reaction between cerulenin and yeast FAS, and between iodoacetamide and FAS, respectively. These data indicate that yeast FAS reacts with cerulenin about 90 times faster than with iodoacetamide [7].We have already demonstrated that cerulenin reacts with the cysteine thiol group at C2 to give the adduct 2b (Fig. 1) in a model reaction between cerulenin and cysteine methyl

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“…In addition to the catalytic domains, almost half of the yeast FAS molecular mass is contributed by six structural domains, two in the α-subunit (SD1-2α) and four in the β-subunit (SD1-4β). Previous studies (15) have shown that the reaction chambers work independently of one another.…”

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confidence: 98%

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Gipson

Mills

Wouts

et al. 2010

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

126

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Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a threedimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.cryoelectron microscopy | fatty acid synthesis | acyl carrier protein | molecular architecture

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Yeast fatty acid synthase: structure to function relationship

Cited by 44 publications

References 13 publications

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Quaternary structure of human fatty acid synthase by electron cryomicroscopy

Effect of side‐chain structure on inhibition of yeast fatty‐acid synthase by cerulenin analogues

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

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