Mapping of functional interactions between domains of the animal fatty acid synthase by mutant complementation in vitro.
Polypeptides of the animal fatty acid synthase (FAS) consist of three amino-terminal catalytic domains, beta-ketoacyl synthase, malonyl/acetyltransacylase, and dehydrase, separated by a 600-residue structural core from four carboxyl-terminal catalytic domains, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase. In the active dimeric form of the protein the two identical multifunctional polypeptides are oriented head-to-tail such that two sites for palmitate synthesis are formed at the subunit interface. In order to map the functional interactions between domains of the two subunits that contribute to the two sites of synthesis, we have utilized a strategy based on complementation analysis in vitro of modified FASs carrying mutations in specific catalytic domains. Homodimeric mutant FASs lacking functional beta-ketoacyl synthase (KS-), dehydrase (DH-), acyl carrier protein (ACP-), or thioesterase (TE-) domains, as well as heterodimers formed between ACP- and TE- subunits, between ACP- and DH- subunits, and between DH- and TE- subunits, were unable to synthesize fatty acids. However, heterodimers formed between KS- and either DH-, ACP-, or TE- subunits regained partial FAS activity. These data indicate that the dehydrase domain, although located in the amino-terminal half of the polypeptide, should be assigned to the complementation group located in the carboxy-terminal half that includes the acyl carrier protein and thioesterase domains. Thus, the current model for the animal FAS must be revised to reflect the finding that the two constituent polypeptides are not simply positioned side-by-side in a fully extended conformation but are coiled in a manner that allows the dehydrase domain to access the beta-hydroxyacyl-ACP located more than 1100 residues distant on the same subunit.
Joshi
A K
AK
Children's Hospital Oakland Research Institute, California 94609, USA.
Witkowski
A
A
Smith
S
S
D000818:Animals; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010449:Peptide Mapping
D064429:Fatty Acid Synthases