Biochhnica et Biophysica Acta, 1130 ( Iq92) 151 - 159 151 c~,; 1992 Elsevier Science Publishers B.V. All rights reserved 01~7-4838/92/505.11{1 BBAPRO 34151) 3-Oxoacyl-[ACP] reductase from oilseed rape ( Brassica napus) Philip S. Sheldon ", Roy G.O. Kekwick :', Colin G. Smith b, Christopher Sidebottom h and Antoni R. Slabas b.I Department of Bu~'hemi.~tO', Unircrsio"of Birmingham, Edgbaston. Birmingham ( UK) and h Biosciences Dirision, Unih'~'erResearch. ('ohvorth lhm.~,. Sharnbr~u~k. Bedford (UK) (Received 18 September 1991) Key words: 3-Oxoacvl-lACPi reducta.~e: Oilseed rape; Fatty acid synthesis: Nt~lulation gent: (B. napus) 3-Oxoacyl-[ACP] reductasc (E.C.I.l.l.ll~J, alternatively known as /3-kctoacyl-[ACP] rcductase), a component of fatty acid synthetase has been purifed frt~m seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high pcrfi~rmance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-31) kDa eoml~ments and has a strong tendcncy to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reducta~ both indicate that the multiple coml~ments have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (K m = 3/zM) twer the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 raM) and CoA (Km = 261 tzM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside thc plastids of rape embryo and leaf tissues by immunoelectron microcopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed string similarities with 3-oxoacyl-[ACP] reductasc from avocado pear and the Nod G gent product from Rhizobium meliloti. Introduction in plants de novo fatty acid synthesis is iocaliscd within the plastids and is catalysed by a system analo- gous to that of E. coli, in which all the components are carried on separate polypeptide chains (reviewed in Ref. I). 3-Oxoaeyl-[ACP] reductase (E.C. 1.1.1.100) catalyses the first reduction step in the fatty acid syn- thesis cycle. It has been purified to homogeneity from spinach leaf [2] and avocado pears [3] and has al~ been characterised in safflower seed [4], Euglena [5], and barley leaf [6]. In oil rich tissues such as some fruits and seeds, triacylglycerol is the predominant end product made from newly synthesised fatty acids, in order to meet this demand, the levels of enzymes involved in the fatty acid synthesis pathway become elevated specifically in these tissues [1,7-9]. In sccds there is evidence that acyl carrier protein can exist in a different form from that of its counterpart in leaf [10]. It is therefore conceivable that this will also be the case for the other components of the syntheta~. In this paper we describe the complete purification of 3-oxoacyl-[ACP] reductase for the first time from any seed tissue, its catalytic properties and subcellular Iocalisation by immunoclectron microscopy. Materials and Methods n Present address: Department of Biological Sciences. University of Durham, Science Buildings South Road. Durham, Dill 3LE, UK. Abbreviations: ACP, acyl carrier protein. Correspondence: P.A. Sheldon. Present address: Department of Biochemistry and Molecular Biology, University of Leeds. Leeds LS2 9JT, UK. Materials The biotinylated donkey anti-rabbit igG and horseradish peroxidase-streptavidin complex were from Amersham International Pie. (Rickmansworth, UK). Staphylococcus V8 protease was from Boehringer Ltd. (Mannheim, Germany). The Aquapore RP-300 column was from Anachem (Luton, UK). The sources of other materials are described in Ref. 3.