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...

Human fatty acid synthase (hFAS) is a homodimeric multidomain enzyme that catalyzes a series of reactions leading to the de novo biosynthesis of long-chain fatty acids, mainly palmitate. The carboxy-terminal thioesterase (TE) domain determines the length of the fatty acyl chain and its ultimate release by hydrolysis. Because of the upregulation of hFAS in a variety of cancers, it is a target for antiproliferative agent development. Dietary long-chain polyunsaturated fatty acids (PUFAs) have been known to confer beneficial effects on many diseases and health conditions, including cancers, inflammations, diabetes, and heart diseases, but the precise molecular mechanisms involved have not been elucidated. We report the 1.48 Å crystal structure of the hFAS TE domain covalently modified and inactivated by methyl γ-linolenylfluorophosphonate. Whereas the structure confirmed the phosphorylation by the phosphonate head group of the active site serine, it also unexpectedly revealed the binding of the 18-carbon polyunsaturated γ-linolenyl tail in a long groove-tunnel site, which itself is formed mainly by the emergence of an α helix (the "helix flap"). We then found inhibition of the TE domain activity by the PUFA dihomo-γ-linolenic acid; γ-and α-linolenic acids, two popular dietary PUFAs, were less effective. Dihomo-γ-linolenic acid also inhibited fatty acid biosynthesis in 3T3-L1 preadipocytes and selective human breast cancer cell lines, including SKBR3 and MDAMB231. In addition to revealing a novel mechanism for the molecular recognition of a polyunsaturated fatty acyl chain, our results offer a new framework for developing potent FAS inhibitors as therapeutics against cancers and other diseases.X-ray crystallography | conformational change | fatty acid selectivity | two-subdomain binding site

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