Abstract
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.
Highlights
SpS cells using a baculoviral vectoTr.he mutated recom- cent to the enoyl reductase domain, this claim has not been binant fattyacid synthase retained all partial activities substantiated by amino-terminal sequencing of the polypeptide of the multifunctional complex except the dehydrase fragment
Hise7*in the dehydration reaction and establishes that the dehydrase domain is located immediately adjacent to the carboxyl terminusof the transferase domain.Examination of the completed domainmap for the animal fatty acid synthaseindicates that the catalytic domains are clusteredin two groups separatebdy a central strucacid sequence and in the ordering of the constituent domains [11,12]
(18).In order tominimize the possibility of inadvertent intro- when the overall fatty acid synthase activity was assessed raduction of additional mutations, we transferred only a small diochemically, by measuring the incorporation of [2-14Clmalofragment (203 bp) of the PCR-amplified region into the final nyl-CoAinto fattyacid, no radioactivity was extracted from the construct
Summary
Materials-The sources of materials have been described in detail as acceptor, by determiningspectrophotometrically the free CoA reelsewhere [18].Plasmids pFAS13.20 and pFAS305.20 were constructed leased in a coupled ATPcitrate-lyasdmalate dehydrogenase reaction by inserting thePCR amplified EcoRI-SphI andEcoRI-Not fragments, [7]. Plasmid pFAS203 consists of the full-length fattyacid synthase cDNA mM NADPH, enzyme, and 10mM trans-1-decalone as substrate. Thioesterase activity was assessed radiochemicallyby extracting tional protein in insect Sf9 cells using baculoviral vectors has been and assaying the [l4C1palmitic acid formed from [l-'4C]palmitoyl-CoA described elsewhere[18].The H878A mutation was introduced into the during anincubation of 3-min duration [24]; assay systems contained in fatty acid synthase using the polymerase chain reaction, as follows. A a final volumeof 0.1 ml, 25 mM potassium phosphate buffer (pH81, 20 portion of thetemplate DNA, pFAS305.20, wasamplifiedusinga pg/ml bovine serum albumin, 10 p~[1-14Clpalmitoyl-CoA(20 nCi), and primer encoding the H878A mutation (FAS878T) in combination with enzyme.
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