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Fatty acid synthase: in vitro complementation of inactive mutants
A Witkowski1, A Joshi, S Smith
1Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Biochemistry
|August 13, 1996
Summary
Researchers created functional fatty acid synthase (FAS) heterodimers from inactive mutants. This study reveals crucial inter-subunit communications within the FAS complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Animal fatty acid synthase (FAS) is a large, multifunctional enzyme complex.
- FAS functions as an antiparallel dimer, with two catalytic centers at the subunit interface.
- Understanding interdomain and intersubunit communication is vital for FAS operation.
Purpose of the Study:
- To investigate interdomain and intersubunit communications in fatty acid synthase.
- To explore the reassembly of catalytically active heterodimers from inactive FAS mutants.
Main Methods:
- Engineered rat fatty acid synthase (FAS) mutants defective in beta-ketoacyl synthase (KS-FAS) or acyl carrier protein (ACP-FAS) domains.
- Expressed and purified mutants using insect Sf9 cells and a baculovirus system.
- Developed a procedure for producing and isolating mixed mutant dimers.
Main Results:
- Homodimeric mutants and KS-FAS heterodimers were catalytically inactive.
- KS-FAS/ACP-FAS heterodimers regained partial fatty acid synthesis activity.
- Heterodimer formation required dissociation and reassociation, indicating negligible spontaneous subunit exchange.
Conclusions:
- Catalytically active heterodimers can be formed from inactive, complementary homodimers.
- This method validates the current model of the multifunctional fatty acid synthase complex.
- Essential residues C161, K326, and S2151A were confirmed for FAS activity.