Construction, expression, and characterization of a mutated animal fatty acid synthase deficient in the dehydrase function.
The location of the dehydrase domain in the multifunctional animal fatty acid synthase has been determined by engineering a fatty acid synthase mutant deficient in dehydrase activity. A full-length fatty acid synthase cDNA encoding a mutated histidine residue (His878-->Ala) was constructed and expressed in insect Sf9 cells using a baculoviral vector. The mutated recombinant fatty acid synthase retained all partial activities of the multifunctional complex except the dehydrase and was unable to synthesize fatty acids. beta-Hydroxybutyryl moieties were formed by the mutant fatty acid synthase from acetyl-CoA, malonyl-CoA, and NADPH and slowly released as the CoA thioester, confirming that this protein cannot perform the dehydration reaction. This finding points to an important catalytic role for His878 in the dehydration reaction and establishes that the dehydrase domain is located immediately adjacent to the carboxyl terminus of the transferase domain. Examination of the completed domain map for the animal fatty acid synthase indicates that the catalytic domains are clustered in two groups separated by a central structural core: the ketoacyl synthase, malonyl/acetyltransferase, and dehydrase in the amino-terminal half and the enoyl reductase, ketoreductase, acyl carrier protein, and thioesterase in the carboxyl-terminal half. A model is proposed in which the two centers for acyl chain initiation, elongation and termination, are formed by the cooperation of the three amino-terminal domains of one subunit with the four carboxyl-terminal domains of the other subunit.
Joshi
A K
AK
Children's Hospital Oakland Research Institute, California 94609.
Smith
S
S
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D064429:Fatty Acid Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D064429:Fatty Acid Synthases; D006836:Hydro-Lyases; D000409:Alanine