Amino acid sequence analysis of rape seed (Brassica napus) NADH-enoyl ACP reductase

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Plant Molecular Biology 17:911-914, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.

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Update section Sequence

Amino acid sequence analysis of rape seed NADH-enoyl ACP reductase

(Brassica napus)

Antoni R. Slabas 1, Ian Cottingham 2, Andrew Austin 2, Tony Fawcett I and Christopher M. Sidebottom 2 l Plant Molecular Biology Group, University of Durham, Durham DH1 3LE, UK; 2 Cell Sciences Section, Unilever Research, Colworth House, Sharnbrook, Bedford, MK44 1LQ, UK Received 21 May 1991; accepted 24 May 1991

The biosynthesis of fatty acids in plants is catalysed by a type II, dissociable fatty acid synthetase (for review see [ 15]. This enzyme system contains at least seven catalytic domains and a central acyl carrier protein (ACP). The individual catalytic reactions are performed on ACP substrates [6] to which the acyl groups are attached via the 4phosphopantetheine group of the protein. Much is known about the structure of ACP in plants. c D N A [8, 9] and genomic clones have been isolated [3, 5, 7] and the tissue-specific and temporal regulation of the gene has been reported [3]. However, comparatively little is known concerning the structure of catalytic components of the plant fatty acid synthetase complex. Two reductive steps occur in core fatty acid biosynthesis, which are catalysed by enoyl ACP reductase and fl-ketoacyl ACP reductase. Enoyl ACP reductase catalyses the second reductive step in fatty acid biosynthesis and there are two forms of this enzyme, a N A D H and a N A D P H enzyme. These are termed type I and type II respectively, and the activities have been separated in Safflower seed [10]. Whilst the type I and type II enzymes are present in seed material, only type I is present in leaf material [ 11 ]. The activ-

ity of the type I enoyl ACP reductase has been measured during seed development in oil seed rape. The activity rises prior to major storage lipid synthesis and the shape of the activity profile closely resembles that of lipid deposition [12]. Western blotting has demonstrated that the protein is continually synthesized during lipid deposition, presumably to meet the high demand for fatty acid synthesis in that part of seed development [ 14]. The enzyme is tetrameric and consists of four identical subunits of 33.6 k D a [ 14] . The rape enzyme has two arginine residues which are covalently modified by phenylyglyoxal, following which the enzyme is inactivated. Such inactivation can be protected by pre-incubation with ACP or coenzyme A, indicating that the arginine residues are either at the active site of the enzyme or that substrate binding results in a conformational change which affords protection [2]. This interaction with ACP has been demonstrated further by affinity chromatography of enoyl ACP reductase on an ACP-sepharose column. In this report, we present extensive amino acid sequence data derived from homogeneous NADH-specific enoyl ACP reductase from oil seed rape. Homogeneous 0c-4 NADH-specific enoyl ACP

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number P80030.

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Fig. 1. Reversed-phase HPLC separation of CNBr (a), tryp-

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tic digestion 1 (b) and tryptic digestion 2 (c) of enoyl ACP reductase. The peptides were separated on a Brownlee C8 reversed-phase 300A column, a, 2.1 x 3 0 m m ; b, c, 1 x 250 mm. Both columns were run at 70 I~l/min and the gradients developed with the following buffers: A = 0 . 1 % TFA; B = 90 % acetonitrile/0.1% TFA.

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