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Structural and Functional Analysis of the Thioesterase Domain of Human Fatty Acid Synthase

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abstract
Human fatty acid synthase (FAS) has recently become one of the most therapeutically relevant targets in the cell. Because of the strong correlation between the elevated expression levels of FAS and most aggressive carcinomas, FAS harbors extreme potential for the treatment of a broad range of human cancers. Recent advances with pharmacological inhibition of FAS have shown promising anti-tumor activity. The FDA-approved drug Orlistat was discovered as a potent inhibitor of the thioesterase activity, which is the seventh functional domain of the multienzyme FAS assembly. Responsible for the highly selective hydrolysis of palmitate (C16) and stearate (C18), thus termination of the fatty acid biosynthetic cycle, inhibition of thioesterase by Orlistat induces tumor cell-specific apoptosis, inhibits the growth of prostate tumors and prevents angiogenesis. This prompted the recent crystal structure of the thioesterase domain inhibited by Orlistat. These complexes confirmed the mechanism of Orlistat mediated inhibition and characterized the active site ‘molecular landscape.’ Thus, providing a foundation for the development of novel anti-tumor agents with the potential to translate into the clinical oncology setting. Futhermore, the binding modes of the Orlistat molecules have enabled a model to be proposed for chain-length selectivity of the thioesterase during the FAS catalytic cycle. These data have driven the design of additional experiments to test hypotheses concerning the mechanism of chain-length selectivity and the biochemistry of ACP mediated substrate delivery and potential domain interactions. In this regard, considerable effort has been dedicated to understanding the highly selective mechanism of C16 and C18 chain-length hydrolysis catalyzed by the thioesterase domain. We have solved to high-resolution three crystal structures of the thioesterase domain in complex with the intact palmitoyl-coenzyme A (CoA) substrate, the double products (CoASH and palmitate), or the single palmitoyl-product. The phosphopantetheinyl arm interacts with a prominent channel suggesting a mechanism for the efficient delivery of acyl-substrates. Moreover, the binding mode of the C16 acyl-chains bound to the hydrophobic ‘specificity channel’ and not the ‘interface cavity’ supports a revised model for the discrimination of acyl-chain length during the FAS catalytic cycle. Mutagenesis designed to fill the interface cavity retained chain-length selectivity nearly identical to wild-type, whereas truncating or lengthening the specificity channel either completely abolished specificity toward C16-18 substrates shifting selectivity toward short- to medium-chain fatty acids (C8-12) or resulted in a notable trend toward longer-chain (≥C18) specificity, respectively. Our observations also provide a foundation for understanding the potential domain interactions through molecular modeling of the docked ACP onto the thioesterase.
subject
chain-length selectivity
Fatty acid synthase
Orlistat
thioesterase
contributor
Pemble, Charles Williford (author)
cpemble@csb.wfu.edu (authorEmail)
Steven J. Kridel (committee chair)
Douglas S. Lyles (committee member)
Thomas Hollis (committee member)
Al Claiborne (committee member)
W. Todd Lowther (committee member)
creator
Pemble, Charles Williford
date
2008-09-28T10:50:47Z (accessioned)
2010-06-18T18:59:17Z (accessioned)
null (available)
2008-09-28T10:50:47Z (available)
2010-06-18T18:59:17Z (available)
2008-08-14 (issued)
degree
null (defenseDate)
Biochemistry & Molecular Biology (discipline)
Wake Forest University (grantor)
PHD (level)
identifier
pemblecw_08_2008.pdf
http://hdl.handle.net/10339/14842 (uri)
migration
etd-08192008-130747 (oldETDId)
rights
Release the entire work for access only to the Wake Forest University system for one year from the date of approval. After one year, release the entire work for access worldwide, unless I send notification to delay release. (accessRights)
I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Wake Forest University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. (license)
title
Structural and Functional Analysis of the Thioesterase Domain of Human Fatty Acid Synthase

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