Structure of fatty acid synthetase from the harderian gland of guinea pig

Kitamoto, Toshihiro; Nishigai, Masaaki; Sasaki, Takuji; Ikai, Atsushi

doi:10.1016/0022-2836(88)90101-5

Cited by 23 publications

(22 citation statements)

References 31 publications

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“…Although the 20-Å cryo-EM reconstruction structure of human FAS dimer has been described (31), there was no indication of the N-to C-terminal polarity of the FAS subunit except for a previous indication from antibody labeling that the FAS TE domain was located at one of the ends of the entire structure (42). We attempted a preliminary assessment of the polarity by manually docking the smaller (32-kDa) human FAS TE domain structure, which occupies the Cterminal end of FAS subunit, and the larger (42 kDa) E. coli ␤-ketoacyl synthase I (or KSI) crystal structure (PDB ID code 1ek4) (43), which is a close homolog of the N-terminal KS domain of mammalian FAS, into each end of the monomer unit designated head and foot of the cryo-EM mass density (Fig.…”

Section: Resultsmentioning

confidence: 99%

Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

Chakravarty

Chirala

et al. 2004

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

179

200

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Human fatty acid synthase is a large homodimeric multifunctional enzyme that synthesizes palmitic acid. The unique carboxyl terminal thioesterase domain of fatty acid synthase hydrolyzes the growing fatty acid chain and plays a critical role in regulating the chain length of fatty acid released. Also, the up-regulation of human fatty acid synthase in a variety of cancer makes the thioesterase a candidate target for therapeutic treatment. The 2.6-Å resolution structure of human fatty acid synthase thioesterase domain reported here is comprised of two dissimilar subdomains, A and B. The smaller subdomain B is composed entirely of ␣-helices arranged in an atypical fold, whereas the A subdomain is a variation of the ␣͞␤ hydrolase fold. The structure revealed the presence of a hydrophobic groove with a distal pocket at the interface of the two subdomains, which constitutes the candidate substrate binding site. The length and largely hydrophobic nature of the groove and pocket are consistent with the high selectivity of the thioesterase for palmitoyl acyl substrate. The structure also set the identity of the Asp residue of the catalytic triad of Ser, His, and Asp located in subdomain A at the proximal end of the groove. Human fatty acid synthase (FAS) is a complex homodimeric (552-kDa) enzyme that regulates the de novo biosynthesis of long-chain fatty acids. This cytosolic enzyme catalyzes the formation of 16 carbon (C 16 ) palmitate, from acetyl-coenzyme A (acetyl-CoA) and malonyl-coenzyme A (malonyl-CoA) in the presence of NADPH. This entire reaction is composed of numerous sequential reactions and acyl intermediates, each catalyzed by a specific enzyme activity (1, 2). Mammalian FAS not only is an essential enzyme in fatty acid synthesis, but also plays an important role during embryonic development (3). Moreover, human FAS recently gained prominence due to its role in obesity and cancer biology. Obesity is a major health problem in developed nations affecting Ͼ50% of the U.S. population and seems to be increasing both in severity and prevalence (4). The magnitude of this health problem and the recent difficulties with several weight-loss therapies emphasize the need for different approaches to treat this problem. Of all of the lipogenic enzymes in the fatty acid synthesis pathway, FAS provides the best opportunity for therapeutic applications because of its high expression in lipogenic tissues and multistep enzyme reactions.Human FAS is an attractive target as both a diagnostic and a prognostic marker for cancer cells. FAS is present at abnormally elevated levels in many varieties of common human cancer, including those of the breast (5-8), prostate (9, 10), colon (11), endometrium (12), ovary (13), thyroid (14), and skin (15). As an anticancer drug target, potentially each of the activities of FAS can be exploited for structure-based design of therapeutic agents. For example, the inhibition of FAS thioesterase (TE) was recently found to halt tumor cell proliferation and inhibit the growth of prostrate tumors in m...

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Section: Resultsmentioning

confidence: 99%

Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

Chakravarty

Chirala

et al. 2004

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

179

200

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“…The combination of native overexpression in the starting material with the use of high-resolution preparative scale anion exchange and gel filtration chromatography yielded highly purified, monodisperse animal FAS . While fungal FAS is a highly symmetric, mostly rigid particle, animal FAS is a dynamic assembly that exists in several conformations, often without the 2-fold symmetry expected for the dimeric molecule (Kitamoto et al 1988). Therefore, despite its smaller size, animal FAS appears as the more difficult target for crystallization than fungal FAS.…”

Section: Purification and Crystallization Of Animal Fasmentioning

confidence: 99%

Structure and function of eukaryotic fatty acid synthases

Maier

Leibundgut

Boehringer

et al. 2010

Quart. Rev. Biophys.

123

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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“…Recently, high-quality electron-micrographic images of the guinea-pig fatty-acid synthase have been obtained and examined by graphic analysis [44]. The enzyme has overall dimensions of 22 x 15 x 7 nm and comprises two \…”

Section: Structural Organization Of the Animal Fatty-acid Synthasementioning

confidence: 99%

“…The order and location of the functional domains is taken from data presented in this paper The overall shape and dimensions of the dimer were established by electron-microscopic imaging [44] and the distances between active sites of the various domains by fluorescence-resonance-energytransfer measurements [46, 471 ACP, acyl-carrier-protein, KS, oxoacyl synthase, TE, thioesterase, M/AT, malonylacetyl transferase, DH, dehydrase elongated subunits, each composed of three major subregions. The polarity of the images was established using domainspecific antibodies and confirmed that the subunits are arranged head-to-tail and that the central bulge in the threedimensional image of each subunit did indeed correspond to the central subregion in the linear polypeptide.…”

Section: Fig 8 Model Of the Dimeric Animal Fatty-acid Vynthasementioning

confidence: 99%

Structural organization of the multifunctional animal fatty‐acid synthase

Witkowski¹,

Rangan²,

Randhawa

et al. 1991

European Journal of Biochemistry

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The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/ acetyl transferase, residues 1630 -1850 to the enoyl reductase, residues 1870 -2100 to the oxyreductase, residues 21 14-2190 to the acyl-carrier protein and residues 2200 -2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.The de novo synthesis of fatty acids by the fatty-acid synthase requires the participation of several enzyme activities. In most bacteria and in plants, the activities exist as discrete monofunctional polypeptides. However, in fungi the functional components are distributed between two nonidentical polypeptides of 205 130 Da Preliminary mapping of the fatty-acid-synthase domain was based largely on analyses of limited proteolysis data. As amino acid sequence data became available, attempts were made to refine the domain map. However, there is disagreement as to the size of the polypeptide chain and the ordering of some of the domains [4,8, 91. In this paper we have re-evaluated the data pertaining to the exact size of the fatty-acid-synthase subunit and have refined the map by identifying the interdomain boundaries separating the various functional domains. The location of these linker regions has been established using several criteria which, although on their own may not be reliable indicators, collectively should give a good indication as to the boundary positions: (a) identifying si...

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Structure of fatty acid synthetase from the harderian gland of guinea pig

Cited by 23 publications

References 31 publications

Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

Structure and function of eukaryotic fatty acid synthases

Structural organization of the multifunctional animal fatty‐acid synthase

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