Animal fatty acid synthase: Functional mapping and cloning and expression of the domain I constituent activities

AUTOR(ES)

Chirala, Subrahmanyam S.

FONTE

The National Academy of Sciences of the USA

RESUMO

Animal fatty acid synthase (FAS; EC 2.3.1.85) is a homodimer of a multifunctional subunit protein and catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH. The subunit (Mr ≈270,000) carries seven distinct component activities and a site for the prosthetic group 4′-phosphopantetheine (acyl carrier protein). Based on proteolytic mapping, the organization of the activity domains along the subunit polypeptide from the N terminus is as follows: β-ketoacyl synthase, acetyl and malonyl transacylases, β-hydroxyacyl dehydratase, enoyl reductase, β-ketoacyl reductase, acyl carrier protein, and thioesterase. By comparing the amino acid sequences of the chicken, rat, and human synthases, we found that kallikrein cleavage sites occur in the least conserved regions of the FAS polypeptide subunit. Determining the amino acid sequences of the N-terminal end of the major kallikrein cleavage peptides helped delineate the most likely boundaries of the component activities in the cDNA-derived amino acid sequence. To confirm this organization, we cloned the chicken FAS cDNA coding for domain I and expressed it in Escherichia coli as a maltose-binding fusion protein. The isolated recombinant protein contained the activities of the acetyl and malonyl transacylases and the β-hydroxyacyl dehydratase. Based on the boundaries of the acetyl and malonyl transacylases and the β-hydroxyacyl dehydratase, we also cloned the appropriate cDNA fragments encoding the domains that contain the transacylases and the dehydratase in pET vectors and expressed them in E. coli as thioredoxin-6xHis fusion proteins. The purified recombinant proteins contained, respectively, the activities of the acetyl and malonyl transacylases and the dehydratase. These results not only confirmed the order of the component activities in domain I, but also paved the way for successful expression and characterization of the remaining activities.

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