ANIMAL FATTY-ACID SYNTHASE - FUNCTIONAL MAPPING AND CLONING AND EXPRESSION OF THE DOMAIN-I CONSTITUENT ACTIVITIES

Animal fatty acid synthase (FAS; EC 2.3.1.85) is a homodimer of a mult ifunctional subunit protein and catalyzes the synthesis of palmitate f rom acetyl-CoA, malonyl-CoA, and NADPH. The subunit (M-r approximate t o 270,000) carries seven distinct component activities and a site for the prosthetic group 4'-phosphopantetheine (acyl carrier protein), Bas ed on proteolytic mapping, the organization of the activity domains al ong the subunit polypeptide from the N terminus is as follows: beta-ke toacyl synthase, acetyl and malonyl transacylases, beta-hydroxyacyl de hydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier prot ein, and thioesterase, By comparing the amino acid sequences of the ch icken, rat, and human synthases, we found that kallikrein cleavage sit es occur in the least conserved regions of the FAS polypeptide subunit , Determining the amino acid sequences of the N-terminal end of the ma jor kallikrein cleavage peptides helped delineate the most likely boun daries of the component activities in the cDNA-derived amino acid sequ ence, To confirm this organization, we cloned the chicken FAS cDNA cod ing for domain I and expressed it in Escherichia coli as a maltose-bin ding fusion protein, The isolated recombinant protein contained the ac tivities of the acetyl and malonyl transacylases and the beta-hydroxya cyl dehydratase, Based on the boundaries of the acetyl and malonyl tra nsacylases and the beta-hydroxyacyl dehydratase, we also cloned the ap propriate cDNA fragments encoding the domains that contain the transac ylases and the dehydratase in pET vectors and expressed them in E. col i as thioredoxin-6xHis fusion proteins, The purified recombinant prote ins contained, respectively, the activities of the acetyl and malonyl transacylases and the dehydratase, These results not only confirmed th e order of the component activities in domain I, but also paved the wa y for successful expression and characterization of the remaining acti vities.