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

An in vitro mutant complementation approach has been used to map the functi onal topology of the animal fatty acid synthase. A series of knockout mutan ts was engineered, each mutant compromised in one of the seven functional d omains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comp rised of a subunit containing either a beta -ketoacyl synthase or malonl/ac etyltransferase mutant, paired with a subunit containing mutations in any o ne of the other five domains, are active in fatty acid synthesis. Heterodim ers in which both subunits carry a knockout mutation in either the dehydras e, enoyl reductase, keto reductase, or acyl carrier protein are inactive, H eterodimers comprised of a subunit containing a thioesterase mutation paire d with a subunit containing a mutation in either the dehydrase, enoyl reduc tase, beta -ketoacyl reductase, or acyl carrier protein domains exhibit ver y low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensatio n reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta -ketoacyl synthas e domains, respectively, of either subunit. The beta -carbon-processing rea ctions, 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 en oyl reductase domains associated exclusively with the same subunit. The cha in-terminating reaction is carried out most efficiently by cooperation of a n acyl carrier protein domain with the thioesterase domain of the same subu nit. These results are discussed in the context of a revised model for the fatty acid synthase.