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Knockout of the regulatory site of 3‐ketoacyl‐ACP synthase III enhances short‐ and medium‐chain acyl‐ACP synthesis
Author(s) -
Abbadi Amine,
Brummel Monika,
Spener Friedrich
Publication year - 2000
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2000.00841.x
Subject(s) - acyl carrier protein , biochemistry , fatty acid synthase , enzyme , mutant , atp synthase , serine , acetyl coa , fatty acid , acyl coa , biosynthesis , stereochemistry , tetrapeptide , biology , chemistry , gene , peptide
Summary 3‐ketoacyl‐acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl‐acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium‐chain acyl‐ACP end products of the FAS reaction, i.e. inhibition by lauroyl‐ACP was uncompetitive towards acetyl CoA and non‐competitive with regard to malonyl‐ACP. This indicated a distinct attachment site for regulatory acyl‐ACPs. Based on alignment of primary structures of various KAS IIIs and 3‐ketoacyl CoA synthases, we suspected the motif G 290 NTSAAS 296 to be responsible for binding of regulatory acyl‐ACPs. Deletion of the tetrapeptide G 290 NTS 293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine 291 to aspartate, alanine 294 to serine and alanine 295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl‐ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium‐chain fatty acid‐producing Cuphea lanceolata seeds and long‐chain fatty acid‐producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short‐chain acyl‐ACPs in the case of C. lanceolata , and of medium‐chain acyl‐ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non‐transgenic plants for production of seed oils with new qualities.