In animals, the seven enzymes involved in do novo fatty acid synthesis from malonyl-CoA are integrated into two polyfunctional polypeptides. The growing acyl chain is translocated on a mobile 4'-phosphopantetheine arm repeatedly through sites of condensation, ketoreduction, dehydration and enoyl reduction, ultimately to the site of chain termination. The animal fatty acid synthetase offers a unique opportunity for evaluating possible advantages of a polyfunctional polypeptide system of organization over a collection of separate, discrete enzymes, for the chain-terminating reaction can be catalyzed with equal efficiency either by one of the covalently-linked domains, thioesterase I, or by a separate medium-chain-teminating enzyme, thioesterase II (e.g. in mammary and uropygial glands), which is not a structural component of the fatty acid synthetase.
One aim of this program is to map the domain structure of the mammalian fatty acid synthetase both linearly, using limited proteolysis as a dissecting tool and a combination of affinity labels, antibodies and partial enzyme activities as specific domain markers, and spatially, using intra and inter-chain cross-linking. A structural basis will be sought to account for the ability of ruminant (but not non-ruminant) fatty acid synthetases to utilize and synthesize medium-chain acyl-CoAs. A novel approach is described to determine whether the two subunits of the mammalian fatty acid synthetase function symmetrically, i.e. each simultaneously elongating an acyl moiety, or asymmetrically, assembling a single acyl moiety per dimer. The mechanism of action of the two types of chain-terminating enzymes will be compared by exploiting reagents, of both the monofunctional and bifunctional type, which attack specifically either the active site region or a putative interfacing region, which facilitates interaction with the fatty acid synthetase. By analysis for amino acid sequency homology of the medium-chain-terminating thioesterases of mamary and uropygial glands, we will explore the possibility that these modified skin glands may have some common evolutionary ancestry.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK016073-12
Application #
3225504
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
12
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Children's Hospital & Res Ctr at Oakland
Department
Type
DUNS #
City
Oakland
State
CA
Country
United States
Zip Code
94609
Brignole, Edward J; Asturias, Francisco (2010) Single-particle electron microscopy of animal fatty acid synthase describing macromolecular rearrangements that enable catalysis. Methods Enzymol 483:179-202
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Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil et al. (2007) Mechanism and substrate recognition of human holo ACP synthase. Chem Biol 14:1243-53
Joshi, Anil K; Witkowski, Andrzej; Berman, Harvey A et al. (2005) Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase. Biochemistry 44:4100-7
Zhang, Lei; Joshi, Anil K; Hofmann, Jorg et al. (2005) Cloning, expression, and characterization of the human mitochondrial beta-ketoacyl synthase. Complementation of the yeast CEM1 knock-out strain. J Biol Chem 280:12422-9
Asturias, Francisco J; Chadick, James Z; Cheung, Iris K et al. (2005) Structure and molecular organization of mammalian fatty acid synthase. Nat Struct Mol Biol 12:225-32
Najjar, Sonia M; Yang, Yan; Fernstrom, Mats A et al. (2005) Insulin acutely decreases hepatic fatty acid synthase activity. Cell Metab 2:43-53

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