Acetoacyl-acyl carrier protein synthase from avocado: its purification, characterisation and clear resolution from acetyl CoA:ACP transacylase.
beta-ketoacyl-ACP synthetase III (KAS III) has been purified from avocado using a six-step purification procedure. The enzyme, which is cerulenin-insensitive and thiolactomycin-sensitive, was assayed using a partial component reaction: acetyl CoA:ACP transacylase (ACAT) activity. KAS III activity is distinguished from ACAT activity on the basis that the former is highly stimulated by the addition of malonyl CoA in the presence of malonyl-CoA:ACP transacylase, and the latter is not. KAS III and ACAT activity have been separated from each other thus providing the first evidence that these two discrete activities exist in higher plants. Both of these enzymes have been implicated in the initial reactions of fatty acid synthesis. KAS III was purified 134-fold using a combination of PEG precipitation, Fast Q, ammonium sulphate precipitation, Phenyl Sepharose and ACP-affinity chromatography. The enzyme requires Triton X-100 for solubility and is highly salt sensitive. The subunit molecular mass of 37 kDa has been identified by SDS-PAGE. The results of gel filtration analysis are consistent with the native enzyme being homodimeric. The native molecular mass of KAS III is 69 kDa and that of ACAT 18.5 kDa. The enzyme has a pH optimum of 7.0-7.5, which is similar to the pH optimum of the ACAT reaction. The Km for acetyl CoA is 12.5 microM and the Km for malonyl-ACP is 14 microM. Both KAS III and ACAT are sensitive to thiolactomycin inhibition. The results are discussed with respect to the potential role of acetyl CoA:ACP transacylase in plants.
Department of Biological Sciences, University of Durham, UK.
B S
BS
Gulliver
A R
AR
Slabas
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051051:Acyl-Carrier Protein S-Acetyltransferase; D002021:Buffers; D002569:Cerulenin / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008970:Molecular Weight; D013876:Thiophenes / Q000494:pharmacology
D002021:Buffers; D013876:Thiophenes; D002569:Cerulenin; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; C035068:thiolactomycin; D000123:Acetyltransferases; C077402:3-ketoacyl-acyl carrier protein synthase III; D051051:Acyl-Carrier Protein S-Acetyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase