Fatty acid synthase: in vitro complementation of inactive mutants.
The animal fatty acid synthase is a dimer of identical, multifunctional 272 kDa subunits oriented antiparallel such that two centers for fatty acid synthesis are formed at the subunit interface. In order to clarify the interdomain and intersubunit communications necessary for the operation of the two centers, we have explored the possibility of reassembling catalytically-active fatty acid synthase heterodimers from pairs of inactive dimers carrying mutations in different functional domains. To this end, rat fatty acid synthase mutants, defective in either the beta-ketoacyl synthase, C161T or K326A (KS- FAS), or the acyl carrier protein, S2151A (ACP- FAS), domains, were engineered by site-directed mutagenesis, expressed in insect Sf9 cells using a baculovirus expression system, and purified. A novel procedure was devised to facilitate rapid production and isolation of a population of mixed mutant dimers that had undergone randomization of its constituent subunits. Homodimeric mutants (KS- FAS/KS- FAS and ACP- FAS/ACP- FAS) and KS- FAS heterodimers consisting of paired C161T and K326A mutant subunits were unable to synthesize fatty acids, confirming the essential nature of residues C161, K326, and S2151A. However, KS- FAS/ACP- FAS heterodimers regained partial activity. Formation of these heterodimers necessitated prior dissociation and reassociation of the homodimers, indicating that the rate of spontaneous exchange of subunits in the dimer is negligible. The formation of catalytically-active heterodimers from pairs of inactive, complementary homodimers affords a useful method for testing the validity of the current model for the multifunctional complex.
Witkowski
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Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Joshi
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Smith
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S
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000378:metabolism; D000213:Acyl Carrier Protein / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D064429:Fatty Acid Synthases / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D018411:Spodoptera
D000213:Acyl Carrier Protein; D018076:DNA, Complementary; D007527:Isoenzymes; C078182:beta-ketoacyl-acyl carrier protein synthase I; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases