Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

Abstract

An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The {~-ketoacyl-ACP reductase, ~-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Ma-trex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the [~-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractio~ were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.Abbreviations: ACP = acyl carrier protein; DTNB = 5",5-dithiobis(2-nitro benzoic acid); DT1 ~ = 1,4-dithiotreitol; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC = high pressure liquid chromatography; kD = kilodalton; Tricine = N-(Tris(hydroxy-methyl)-methyl)-glycin; SDS = sodium dodecyl sulphate.

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes

“…A heterodimer of the latter's resulting polypeptide synthesizes fatty acids (Smith et al 2003 ). In plant plastids and bacteria, the type II FAS complex, generating mostly up to 16-or 18-carbon fatty acids, is an easily dissociable, multisubunit complex consisting of monofunctional enzymes each encoded by a discrete gene (Høj and Mikkelsen 1982 ;Shimakata and Stumpf 1982a ). This complex is formed by four enzymes a β-ketoacyl-[ACP]synthase (KAS I, II or III), a β-ketoacyl-[ACP]-reductase (KAR), a β-hydroxyacyl-[ACP]-dehydratase (HAD) and an enoyl-[ACP]-reductase (ENR).…”