Structure and molecular organization of mammalian fatty acid synthase

De novo synthesis of fatty acids in the cytosol of animal cells is carried out by the multifunctional, homodimeric fatty acid synthase (FAS). Cryo-EM analysis of single FAS particles imaged under conditions that limit conformational variability, combined with gold labeling of the N termini and structural analysis of the FAS monomers, reveals two coiled monomers in an overlapping arrangement. Comparison of dimeric FAS structures related to different steps in the fatty acid synthesis process indicates that only limited local rearrangements are required for catalytic interaction among different functional domains. Monomer coiling probably contributes to FAS efficiency and provides a structural explanation for the reported activity of a FAS monomer dimerized to a catalytically inactive partner. The new FAS structure provides a new paradigm for understanding the architecture of FAS and the related modular polyketide synthases.     

Fatty acid biosynthesis in actinomycetes

All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multienzyme FAS II system and Corynebacterium species exclusively FAS I. In this review, we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with antimycobacterial properties.     

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.     

Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems.

The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits alpha and beta are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell.     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

Microbial Type I Fatty Acid Synthases (FAS): Major Players in a Network of Cellular FAS Systems

The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits α and β are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell.     

Conjugated linoleic acid (CLA) inhibits fatty acid synthetase activity in vitro

This paper describes the in vitro effect of conjugated linoleic acid (CLA) on fatty acid biosynthesis. Among the rat liver enzymes involved in fatty acid biosynthesis, fatty acid synthetase (FAS) showed the largest activity fluctuation with the types of fatty acids. Of the fatty acids, CLA was the most potent inhibitor of FAS, and the 9c, 11t-rather than the 10t, 12c-isomer showed greater inhibition. CLA also significantly lowered the incorporation of [14C]-acetate into phospholipid in breast cancer cells, supporting the view that CLA inhibits fatty acid biosynthesis through the interaction with FAS.     

A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.     

Inhibitory activity of chlorogenic acid on enzymes involved in the fatty acid synthesis in animals and bacteria

It was found that chlorogenic acid inhibited in vitro animal fatty acid synthase (FAS I) and the ss-ketoacyl-ACP reductase (FabG) from Escherichia coli in a concentration-dependent manner with respective IC50 of 94.8 and 88.1 microM. The results of Lineweaver-Burk plots indicated that chlorogenic acid inhibited competitively the binding of NADPH to FAS I, while left those of acetyl-CoA and malonyl-CoA unaffected. Further kinetic studies showed that chlorogenic acid blocked the activity of FAS I mainly by inhibiting the ss-ketoacyl reductase domain, which catalyzed the same reaction as that done by FabG in the fatty acid synthesis. The ss-ketoacyl reduction reactions accomplished by both FAS I and FabG required nucleotide cofactor, NADPH. Furthermore, the Lineweaver-Burk and Yonetani-Theorell analyses implicated that chlorogenic acid filled competitively in the binding-pocket of NADPH in the ss-ketoacyl reductase domain of FAS I. The similar results were also obtained from the inhibition of FabG by chlorogenic acid. As observed in these results, the inhibitions of FAS I and FabG by chlorogenic acid were highly related to the interference of the inhibitor with NADPH, which was possibly due to the similarity between chlorogenic acid and some portion of NADPH, maybe the section consisting of the two ribose groups.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.     

Mitochondrial fatty acid synthesis,fatty acids and mitochondrial physiology

Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the “classic” cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both β-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Mitochondrial fatty acid synthesis in Trypanosoma brucei

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [(14)C]pyruvate or [(14)C]threonine, either of which is catabolized to [(14)C]acetyl-CoA in the mitochondrion. Although some of the [(14)C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

The short-term regulation of fatty acid synthesis in the rat epididymal adipocytes

Lipogenesis and fatty acid synthetase (FAS) activity of isolated rat adipocytes that were treated with insulin or epinephrine were studied. Insulin stimulated incorporation of radioactivity from D-[U-14C]glucose into CO2, saponifiable and non-saponifiable fractions, whereas epinephrine promoted lipolysis and oxidation of glucose into CO2. Whereas insulin stimulated fatty acid synthesis, epinephrine had no effect. Changes in FAS specific activity of insulin- or epinephrine-treated adipocytes were insignificant and could not account for insulin-stimulated lipogenesis. Rat adipocyte FAS, unlike hepatic FAS, was not subject to short-term regulation by insulin, although fatty acid synthesis showed such a response.     

脂肪酸合酶—特异性肿瘤治疗靶点

脂肪酸合成的产物———脂肪酸为肿瘤生长的物质和能量来源。脂肪酸合成的关键酶———脂肪酸合酶(fattyacidsynthase ,FAS)在肿瘤组织中高表达而在正常组织中含量较低 ,表明FAS可成为肿瘤治疗靶点。现就近年来有关FAS、FAS与肿瘤的关系以及FAS抑制剂等问题的研究进展进行综述。     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0