HUMAN FATTY-ACID SYNTHASE - ASSEMBLING RECOMBINANT HALVES OF THE FATTY-ACID SYNTHASE SUBUNIT PROTEIN RECONSTITUTES ENZYME-ACTIVITY

Our model of the native fatty acid synthase (FAS) depicts it as a dime r of two identical multifunctional proteins (M-r approximate to 272,00 0) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Angstrom of the pantetheinyl-SH of t he second subunit (where the malonyl group is bound), This arrangement generates two active centers for fatty acid synthesis and predicts th at if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site, We cloned, ex pressed, and purified catalytically active thioredoxin (TRX) fusion pr oteins of the NH2-terminal half of the human FAS subunit protein (TRX- hFAS-dI; residues 1-1,297; M-r approximate to 166) and of the C-termin al half (TRX-hFAS-dII-III; residues 1,296-2,504; M-r approximate to 15 5), Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH result ed in the synthesis of long-chain fatty acids, The rate of synthesis w as dependent upon the presence of both recombinant proteins and reache d a constant level when they were present in equivalent amounts, indic ating that the reconstitution of an active fatty acid-synthesizing sit e required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to b e the most abundant with small amounts of palmitate and stearate, poss ibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state, The successful reconstitution of the human FAS activi ty from its domain I and domains II and III fully supports our model f or the structure-function relationship of FAS in animal tissues.