Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), ACC1 (Mr ‫؍‬ 265 kDa) and ACC2 (Mr ‫؍‬ 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.A cetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate substrate that plays a pivotal role in the regulation of fatty acid metabolism. Besides its role in the biosynthesis of long-chain fatty acids (1-3), malonyl-CoA has been implicated in the regulation of the carnitine palmitoyl-CoA shuttle system that is involved in the mitochondrial ␤-oxidation of long-chain fatty acids. In animals, two isoforms of the carboxylase have been identified, ACC1 (M r ϭ 265,000) and ACC2 (M r ϭ 280,000) (4, 5). The two enzymes are encoded by separate genes and display distinct tissue distribution and regulation (6-9). The ACC1 carboxylases are highly expressed in lipogenic tissues, such as liver and adipose, and their levels are regulated transcriptionally while their activities are regulated posttranslationally by phosphorylation͞dephosphorylation of selected serine residues and by allosteric regulation through the action of citrate and palmitoyl-CoA (10-18). Dietary and hormonal states of the animal affect the level and activities of the ACC1 enzymes. A carbohydrate-rich, low-fat diet stimulates the expression and activities of ACC1, whereas starvation and diabetes reduce the ACC1 activities by repressing the expression of the ACC1 gene or by increasing the phosphorylation levels of the ACC1 protein (or both). Treating diabetic animals with insulin increases the activity of the enzyme either by dephosphorylation of the protein or by...

We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4

In animals, liver and white adipose are the main sites for the de novo fatty acid synthesis. Deletion of fatty acid synthase or acetyl-CoA carboxylase (ACC) 1 in mice resulted in embryonic lethality, indicating that the de novo fatty acid synthesis is essential for embryonic development. To understand the importance of de novo fatty acid synthesis and the role of ACC1-produced malonyl-CoA in adult mouse tissues, we generated liver-specific ACC1 knockout (LACC1KO) mice. LACC1KO mice have no obvious health problem under normal feeding conditions. Total ACC activity and malonyl-CoA levels were Ϸ70 -75% lower in liver of LACC1KO mice compared with that of the WT mice. In addition, the livers of LACC1KO mice accumulated 40 -70% less triglycerides. Unexpectedly, when fed fat-free diet for 10 days, there was significant up-regulation of PPAR␥ and several enzymes in the lipogenic pathway in the liver of LACC1KO mice compared with the WT mice. Despite the significant up-regulation of the lipogenic enzymes, including a >2-fold increase in fatty acid synthase mRNA, protein, and activity, there was significant decrease in the de novo fatty acid synthesis and triglyceride accumulation in the liver. However, there were no significant changes in blood glucose and fasting ketone body levels. Hence, reducing cytosolic malonyl-CoA and, therefore, the de novo fatty acid synthesis in the liver, does not affect fatty acid oxidation and glucose homeostasis under lipogenic conditions.Cre-loxP ͉ fatty acid synthesis ͉ tissue-specific knockout I n eukaryotes, acetyl-CoA carboxylase (ACC) is a biotinylated enzyme that catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA. Fatty acid synthase (FAS), the multifunctional enzyme, catalyzes the synthesis of long-chain fatty acid, palmitate, by using acetyl-CoA as a primer, malonylCoA as a two-carbon donor for chain elongation, and NADPH for the reduction reactions. The synthesis of malonyl-CoA is the committed step toward the synthesis of fatty acids (1-5). In addition, malonyl-CoA also plays an important role in the regulation of fatty acid oxidation in the mitochondria as an inhibitor of the carnitine palmitoyl transferase 1, which performs the first step in the transfer of long-chain fatty acyl CoA into mitochondria for their oxidation (6, 7). Hence, malonyl-CoA participates in two opposing pathways, a substrate for fatty acid synthesis and a regulator of fatty acid oxidation. Lately, there are also reports that malonyl-CoA regulates orexigenic responses in hypothalamus (8) and insulin secretion by pancreatic ␤-islets (9, 10). Malonyl-CoA is generated by two isoforms of acetyl-CoA carboxylases, ACC1 and ACC2 of molecular mass 265 kDa and 280 kDa, respectively (11-16). ACC1 is a cytosolic enzyme, and ACC2 is associated with mitochondria (17). Although both isoforms are expressed in various tissues, ACC1 is predominantly expressed in lipogenic tissues such as liver, adipose, and lactating mammary gland, and ACC2 is predominantly expressed in muscle tissues and heart ...

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...