Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro.

Abstract

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.