Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution Vesna Katavic 1 , Elzbieta Mietkiewska 2,3 , Dennis L. Barton 1 , E. Michael Giblin 2 , Darwin W. Reed 2 and David C. Taylor 2 1 Saskatchewan Wheat Pool Agricultural Research and Development, Saskatoon, Canada; 2 National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, Canada, 3 Plant Breeding and Acclimatization Institute, Mlochow Research Center, Poland Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Bras- sica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenyl- alanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain mono- unsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the bio- synthesis of the eicosenoic and erucic acids. Keywords: Brassica; Brassicaceae; Fatty Acid Elongation 1; 3-ketoacyl-CoA synthase; site directed mutagenesis. While de novo fatty acid synthesis occurs in plastids, the synthesis of very long chain monounsaturated fatty acids (VLCMFAs) is located in the cytosol and catalyzed by a membrane-bound fatty acid elongation (FAE) complex on the endoplasmic reticulum. The initial substrate for the elongation is oleic acid (18:1). The elongation of 18:1 involves the sequential addition of C 2 units from malonyl- CoA to a long chain acyl-CoA primer. Each round of elongation involves four enzymatic reactions catalyzed by the FAE complex. The FAE reactions are condensation of malonyl-CoA with a long chain acyl-CoA to give a 3-ketoacyl-CoA, reduction to 3-hydroxyacyl-CoA, dehy- dration to enoyl-CoA and final reduction of the enoyl-CoA resulting in an elongated acyl-CoA [1]. In high erucic acid (HEA) Brassicaceae, a seed-specific fatty acid elongase 1 (FAE1) is the condensing enzyme (3-ketoacyl-CoA synthase) that catalyzes the first of four enzymatic reactions of the FAE complex, resulting in the synthesis of VLCMFAs which are the major constituents of their seed oil. It is assumed that the activities of the three subsequent enzymes crucial for VLCMFA biosynthesis, namely a 3-ketoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrase and enoyl-CoA reductase, are present ubiqui- tously in plants and are common to all microsomal FAE systems. In contrast, the condensing enzymes seem to be differentially expressed and are probably unique to each system. Furthermore, it appears that FAE1 is a rate limiting enzyme for VLCMFA accumulation in seeds [2,3] while the other three enzymes of the elongase complex do not appear to play a role in controlling VLCMFA formation [2]. Millar & Kunst [2] demonstrated that the Arabidopsis FAE1 gene is able to direct the synthesis of VLCFA in yeast. Upon the expression of the Arabidopsis FAE1 coding region under the control of strong galactose-inducible (GAL1) promoter, the transformed yeast cells accumulated 20:1, 22:1 and 24:1 that are not normally present in nontrans- formed yeast cultures. Han et al. [4–6] expressed Arabidopsis and B. napus FAE1 genes in yeast cells and concluded that in addition to 18:1 D9, both elongases are able to elongate the 16:1 D9acyl chain. However, the Arabidopsis FAE1 prefers to use 18:1 D9 and 18:1 D11 to produce 20:1 D11 and 20:1 D13, respectively, while the Brassica napus FAE1 more efficiently Correspondence to V. Katavic, NRC/PBI, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada. Fax: + 1 306 975 4839, Tel.: + 1 306 975 5273, E-mail: Vesna.Katavic@nrc.ca Abbreviations: FAE1, fatty acid elongation 1; FAE1, fatty acid elongase 1; FAMEs, fatty acid methyl esters; HEA, high erucic acid; LEA, low erucic acid; LPAT, lyso-phosphatidic acid acyltransferase; 3-KCS, 3-ketoacyl-CoA synthase; MDE, microspore derived embryo; SC-ura, synthetic complete medium lacking uracil; SDM, site directed mutagenesis; VLCMFAs, very long chain monounsaturated fatty acids; VLCFAs, very long chain fatty acids; WS-SDM, Westar culti- var site directed mutated; WS-wt, Westar cultivar wild-type. Note: The nucleotide sequence data reported are deposited in the GenBank database under accession numbers AF490459, AF490460, AF490461 and AF490462. (Received 3 July 2002, revised 21 August 2002, accepted 18 September 2002) Eur. J. Biochem. 269, 5625–5631 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03270.x utilizes 20:1 D11 and 20:1 D13 to make 22:1 D13 and 22:1 D15, respectively. As a part of our effort to increase the amounts of industrially valuable VLCFAs, particularly erucic acid (22:1) in Canadian HEA cultivars, we focused our research on manipulating genes/enzymes which are involved in the accumulation of VLCFAs in seed oil: an erucoyl-CoA- utilizing lyso-phosphatidic acid acyltransferase (LPAT) crucial for trierucin bioassembly, and the seed-specific FAE1, crucial for erucic acid biosynthesis. Earlier, we reported on the expression of yeast LPAT gene SLC1-1 and Arabidopsis FAE1 coding regions in target HEA B. napus germplasm, and the performance of transgenic progeny in the field [3,7,8]. Although the cloning of genes encoding 3-ketoacyl-CoA synthase (3-KCS) from different plant species has been achieved [4,9–21], knowledge about the mechanism of action and properties of FAE1 and other elongase conden- sing enzymes is limited due to the fact that these enzymes are membrane-bound, and as such are inherently more difficult to characterize biochemically than soluble condensing enzymes. Recently, Ghanevati & Jaworski [22] generated and analyzed a number of Arabidopsis FAE1 mutants to decipher the importance of cysteine and histidine residues as possible catalytic residues of FAE1 condensing enzymes. Their results have shown that cysteine 223 is essential for FAE1 KCS activity, and that it seems to have a similar role to the active-site cysteines present in other condensing enzymes. To get more insight into how the coding region of the FAE1 condensing enzymes in Brassicas determines the proportions and amounts of VLCFAs in their seed oils, we have expressed FAE1 coding regions from HEA B. napus, Brassica rapa (formerly Brassica campestris)andBrassica oleracea in yeast. Intense research is ongoing by several groups to elucidate the mutations involved in the loss of FAE1 condensing enzyme activity in LEA B. napus cultivars. Han et al.[6] speculated that the presence of serine at position 282 in all functional proteins instead of phenylalanine in nonfunc- tional LEA B. napus FAE1 could be important for the activity of the condensing enzyme. Roscoe et al.[23] hypothesized that the LEA phenotype could be the result of one or more lesions in the genes that encode or regulate FAE1 activity. To clarify this controversy, we decided to examine the role of the amino acid serine at position 282 in the FAE1 protein sequence to determine if this apparent mutation from serine to phenylalanine led to the LEA B. napus phenotype. We introduced a point mutation into the LEA B. napus cv. Westar FAE1 coding region to substitute phenylalanine with serine at position 282 in an attempt to restore the FAE1 condensing enzyme activity. Here we report and discuss the results of analyses of heterologous expression in yeast and site-directed mutation of Brassica FAE1 condensing enzymes. MATERIALS AND METHODS Plant materials HEA B. napus cv. Hero [24], B. rapa microspore-derived embryo line, MDE R500 [25], B. oleracea microspore- derived embryo line, MDE 103 [26], and LEA B. napus canola cv. Westar were used in this study for the cloning of FAE1 coding regions. Cloning FAE1 coding regions and heterologous expression in yeast Based on known FAE1 sequences from Arabidopsis and B. napus, the forward primer VBE4 (5¢-ACCATG ACGTCCATTAACGTAAAGCTCC-3¢) and the reverse primer VBE3 (5¢-GGACCGACCGTTTTGGGCACG-3¢) were designed, synthesized and used to amplify FAE1 coding regions from target species by PCR. Genomic DNA was isolated according to Edwards et al. [27] from seed at mid- development from B. napus cv. Hero and cv. Westar, and from MDEs at mid-development from B. rapa and B. oler- acea. This was used as template DNA for PCR, carried out using Vent DNA polymerase (New England Biolabs). Amplified products without stop codons were cloned into the yeast expression vector pYES2.1/V5-His-TOPO (Invi- trogen) downstream of the galactose-inducible promoter (GAL1). Omitting a stop codon allows for the PCR product to be expressed as a fusion to the C-terminal V5 epitope and polyhistidine tag for protein detection and purification. All products were confirmed by sequence analyses using external primer Gal1 Forward primer (Invitrogen) and V5 C- terminus Reverse primer (Invitrogen), and primers VBE3 and VBE4. Yeast cells (line Inv Sc1, Invitrogen), were transformed with pYES2.1/V5-His-TOPO constructs bear- ing different FAE1 genes, using the S.c. EasyComp TM Transformation Kit (Invitrogen). Yeast cells transformed with pYES2.1/V5-His-TOPO plasmid only were used as a control. Transformants were selected by growth on synthetic complete medium lacking uracil (SC-ura), supplemented with 2% (w/v) glucose. The colonies were transferred into liquid SC-ura with 2% (w/v) glucose and grown at 28 °C overnight. For expression studies the overnight cultures were used to inoculate 25 mL of SC-ura supplemented with 2% (w/v) galactose to give an initial D 600 of 0.2. The cultures were subsequently grown overnight at 20 °Cor28°CtoD 600 of 1.4 and used for biochemical analyses. Fatty acid analyses and enzyme assays The yeast cultures were grown overnight and cells were pelleted. Cell pellets were saponified in methanolic-KOH [10% (w/v) KOH, 5% (v/v) H 2 Oinmethanol]for2hat 80 °C. After saponification, samples were cooled on ice and then washed with hexane to remove nonsaponifiable material. The remaining aqueous phase was then acidified with 6 M HCl. Free fatty acids were extracted in hexane, the solvent removed under a stream of N 2 ,andthefreefatty acids were transmethylated in 3 M methanolic HCl for 2 h at 80 °C. Fatty acid methyl esters (FAMEs) were extracted in hexane, the solvent removed under a N 2 stream and the residue was dissolved in hexane for GC under the conditions described previously [28]. Fatty acid elongase activity of the yeast microsomal membrane preparation was assayed essentially as described by Katavic et al. [3]. The assay mixture consisted of 80 m M Hepes-NaOH, pH 7.2; 1 m M ATP, 1 m M CoA-SH, 0.5 m M NADH, 0.5 m M NADPH, 2 m M MgCl 2 ,1m M malonyl-CoA, 18l M [1– 14 C]oleoyl-CoA (0.37 GBqÆmol )1 ) in a final volume of 500 lL. The reaction was started by 5626 V. Katavic et al.(Eur. J. Biochem. 269) Ó FEBS 2002 the addition of 0.5 mg of microsomal protein and incubated at 30 °C for 1 h. Reactions were stopped by adding 3 mL of 100 gÆL )1 KOH in methanol. FAMEs were prepared and quantified by radio-HPLC as described by Katavic et al.[3]. Site-directed mutagenesis To introduce the desired point mutation into the FAE1 coding region isolated from LEA B. napus cv. Westar, we used a QuikChange TM site-directed mutagenesis kit (Strat- agene). We have designed the oligonucleotide primers SDF-3 (5¢-TGTTGGTGGGGCCGCTATTTTGCTCT CCAACAAG-3¢) and SDF-4 (5¢-CTTGTTGGAGAGC AAAATAGCGGCCCCACCAACA-3¢) containing the desired mutation (bold). Primers were complementary to opposite strands of pYES2.1/V5-His-TOPO containing the FAE1 gene. During the PCR, primers were extended with PfuTurbo DNA polymerase. This polymerase replicated both strands with high fidelity and without displacing the mutated oligonucleotide primers. PCR incubations were run 30 sec at 95 °C (denaturation) followed by 16 cycles of 30 sec at 95 °C, 1 min at 55 °C, 15 min at 68 °Cand terminated by 15 min at 68 °C. Following temperature cycling, the product was treated with Dpn1 endonuclease (target sequence 5¢-Gm 6 ATC-3¢) which is specific for methylated and hemimethylated DNA, and is used to digest parental DNA template and to select mutation- containing synthesized DNA. Microsomal membrane preparation Yeast microsomes were prepared essentially according to Ghanevati & Jaworski [22]. Briefly, cells were harvested and washed with 10 mL of ice-cold isolation buffer (IB, 80 m M Hepes-NaOH, pH 7.2, 5 m M EGTA, 5 m M EDTA, 10 m M KCl, 320 m M sucrose, 2 m M dithiothreitol), pelleted and resuspended in 500 lL of IB. Cells were broken using three 60 s pulses with the Mini-Beadbeater TM (BioSpec Products, Inc., Bartlesville, OK, USA) using 0.5 mm glass beads. The supernatant was collected and centrifuged briefly to remove unbroken cells. The microsomal membrane pellet was recovered after centrifugation at 100 000 g for 60 min and resuspended in IB containing 20% (v/v) glycerol. Protein concentration was determined using the method according to Bradford [29]. Immunoblot analysis Microsomal proteins (100 lg) were separated on 15% SDS/ PAGE Ready Gel (Bio-Rad). After electrophoresis, proteins were electro-transferred (1.5 h, 180 mA, 4 °C) to poly(vinylidene difluoride) (PVDF) membrane (Hybond TM -P, Amersham) using a Mini Trans-blot (Bio-Rad) apparatus and transfer buffer [10 m M CAPS, 10% (v/v) methanol, pH 11.0]. An anti-(FAE1 3-KCS) Ig (giftfromDrL.Kunst,DepartmentofBotany,University of British Columbia, Canada) was used at a dilution of 1 : 5000. Secondary antibody (horseradish peroxidase- linked anti-rabbit IgG from sheep, Amersham) was diluted 1 : 10 000 and detected using Western blotting together with the ECL Plus system (Amersham) and Super RX film (Fujifilm). RESULTS Sequence alignment of Brassica FAE1 proteins We isolated by PCR clones corresponding to the coding regions of FAE1 genes from HEA B. napus cv. Hero, HEA B. rapa line R500, HEA B. oleracea line 103 and LEA B. napus cv. Westar. Nucleotide sequences corres- ponding to open reading frames of 1523 bp were trans- lated and proteins of 506 amino acids were deduced. Aligned FAE1 proteins from different HEA Brassica species showed high homology to published B. napus FAE1 protein sequences (GenBank coding region acces- sion numbers AF006563, cv. Golden, and AF274750, cv. Ascari). However, several unique differences among FAE1 protein sequences were observed. HEA B. napus cv. Hero FAE1 has two unique differences, one at position 118 with asparagine instead of aspartic acid, while at the position 484 in Hero, aspartic acid is substituted by a glutamic acid residue. HEA B. rapa FAE1 has a serine residue at position 179 while all other aligned FAE1 proteins have asparagine residues at this position. When we compared FAE1 protein sequences from different HEA Brassica spp. with the LEA B. napus cv. Westar FAE1, we could detect two unique substitutions in Westar FAE1. At position 282 serine is substituted with phenyl- alanine, and at position 303 threonine is substituted with alanine. However, the alignment of several microsomal 3-KCSs from Arabidopsis thaliana and different Brassic- aceae revealed that the only crucial difference among the protein sequences from functional microsomal 3-KCSs and the nonfunctional FAE1 condensing enzyme from LEA B. napus cv. Westar is at position 282. While all functional elongases have a serine amino acid residue at that position, in the catalytically inactive protein from LEA cv. Westar serine 282 is substituted by phenylalanine (Fig. 1). Fig. 1. Alignment and comparison of amino-acid sequences of several 3-ketoacyl-CoA synthases. Protein sequences spanning the region of amino acids 266–325 from A. thaliana CUT1 (accession number AF129511) and amino acids 264–323 from A. thaliana FAE1 (accession number AF053345), HEA B. napus cv.s Golden and Ascari (accession numbers AF00953 and AF274750), HEA B. napus cv. Hero, B. oleracea MDE line 103, B. rapa MDE line R500 and LEA B. napus cv. Westar were aligned. Amino acid residues at position 282 are shaded in black and indicated by the black arrow. The amino acid residues at position 303 are indicated by the white arrow. Ó FEBS 2002 Restoring enzyme activity in Brassica napus FAE1 (Eur. J. Biochem. 269) 5627 Expression of FAE1 genes in yeast and GC analyses of FAMEs For the functional expression of FAE1 clones in yeast, DNA fragments corresponding to open-reading frames of Brassica FAE1 genes as well as Arabidopsis FAE1 (used as a positive control for FAE1 expression) were linked to GAL1 in the expression vector pYES2.1/V5-His-TOPO. Yeast cells were transformed with FAE1 expression constructs or with expression vector pYES2.1/V5-His-TOPO only (a negative experimental control). Upon expression, the fatty acid composition of induced yeast cell lysates was analyzed by GC. All HEA FAE1 genes were functionally expressed in yeast, and heterologous 3-KCS enzymes together with the endogenous dehydratase and two reductases catalyzed the elongation of long chain fatty acid substrates into VLCFA products. All HEA Brassica FAE1-expressing yeast cells were able to utilize both 18:1 isomers (D9andD11) as substrates for elongation reactions to produce 20:1 D11 and D13 isomers, and 22:1 D13 and D15 isomers, respectively. The relative proportions of the different VLCFAs are shown in Table 1. The A. thaliana FAE1 more poorly utilized 20:1 as a substrate in elongation process compared to FAE1 from HEA Brassica species. The results of expression experiments at two different temperatures (28 °Cvs.20°C) showed that the overall activity of the FAE1 condensing enzyme was reduced at the lower temperature, but the trends in the relative proportions of VLCMFAs produced were similar at both temperatures. Site directed mutagenesis of the LEA B. napus cv. Westar FAE1 gene In order to test the importance of serine 282–3-KCS func- tion, we used a site-directed mutagenesis (SDM) approach to change the phenylalanine 282 residue in LEA cv. Westar FAE1 to the highly conserved serine residue. The sequence analyses of five different clones revealed that two of them (WS-SDM1 and WS-SDM18) had been successfully mutated with a serine at position 282 (data not shown). We expressed wild-type FAE1 (WS-wt), two mutated WS FAE1 clones (WS-SDM1 and WS-SDM18) and the empty plasmid control (pYES2.1/V5-His-TOPO) in yeast cells. The results of fatty acid analyses of transformed yeast cell lysates by GC of the FAMEs revealed that condensing enzyme activity was restored in both mutated WS clones; yeast cells expressing mutated WS-SDM clones produced 20:1 D11 and D13 isomers, and 22:1 D13 and D15 isomers. In contrast, yeast cells expressing WS-wt or plasmid only had fatty acid profiles typical of yeast, with no detectable monounsaturated VLCFAs present (Fig. 2). Immunoblot analyses of yeast microsomes In order to detect FAE1 proteins in yeast cells expressing cv. Westar wild-type FAE1 andtwomutatedcv.WestarFAE1 clones, Western blot analyses were performed using micro- somes isolated from yeast cells after FAE1 heterologous expression and using anti-FAE1 Igs raised against the C-terminus domain of the Arabidopsis FAE1 protein. Protein bands corresponding to FAE1/V5-His fusion were detected in all experimental samples except in the pYES2.1/ V5-His-TOPO-only control (Fig. 3). Elongase activity in microsomal fraction of yeast cells Microsomal fractions were isolated from lysates of yeast cells upon expression of WS-wt FAE1 clone and two mutated clones WS-SDM1 and WS-SDM18. As a control, the microsomal fraction from yeast cells containing only the empty plasmid (pYES2.1/V5-His-TOPO) was used. To analyze the elongase activity, microsomal proteins were incubated with [1- 14 C]18:1-CoA and malonyl CoA. The results of the elongase activity assays are summarized in Table 2. The elongase activity in WS-wt was low as expected, but the activity in the two mutated WS-SDM clones was comparatively very high. DISCUSSION We have isolated genomic clones corresponding to FAE1 coding regions from several HEA Brassica species and from LEA B. napus. No introns were present in the genomic clones, which seems to be a general characteristic of FAE1 genes in Brassicaceae. Our earlier work [3] as well as findings from other groups [2,5,6] have shown that it is the FAE1 3-KCS coding region that determines the preference of its translated protein for either 18:1 moieties or 20:1 moieties for elongation. The A. thaliana FAE1 condensing enzyme used 20:1-CoA more poorly as an elongation substrate Table 1. Fatty acid composition of yeast cells expressing FAE1 condensing enzymes from different HEA Brassica species a . Lysates from yeast cells expressing B. napus cv. Hero, B. oleracea MDE line 103, B. rapa MDE line R500, LEA B. napus cv. Westar FAE1 condensing enzyme were analyzed. Cells expressing FAE1 from A. thaliana (ecotype Columbia) and pYES2.1/V5-His-TOPO (pYES2.1) were used as positive and negative controls, respectively. FA (%), relative percent of total fatty acids. pYES2.1/FAE1 FA (%) 16:0 16:1 18:0 18:1 D9 18:1 D11 20:1 D11 20:1 D13 22:1 D13 22:1 D15 24:1 VLCMFA B. n. Hero 16.34 43.27 3.97 15.15 1.52 0.45 0.99 2.07 1.25 0.15 4.91 B. o. 103 15.77 44.84 3.62 13.90 1.56 0.49 1.10 2.19 1.07 0.17 5.02 B. r. R500 15.22 43.76 3.96 14.97 1.46 0.45 0.94 1.94 1.54 0.46 5.33 B. n. Westar 14.91 45.50 4.75 29.03 1.02 0.07 0.00 0.00 0.00 0.00 0.07 A. t. Col. 16.12 40.64 4.69 18.63 1.68 1.67 3.17 0.85 0.37 0.07 6.13 pYES2.1 19.22 40.67 5.88 24.69 1.01 0.00 0.00 0.00 0.00 0.00 0.00 a The data for 26:0 which is normally present in yeast at the amount of approximately 5%, and other fatty acids (12:0, 14:0, 14:1, 20:0, 22:0, 24:0) which were present in similarly minor percentages in all our samples are not shown. 5628 V. Katavic et al.(Eur. J. Biochem. 269) Ó FEBS 2002 compared to B. napus and its ancestral species B. oleracea and B. rapa. The alignment of FAE1 polypeptides revealed several differences among FAE1 condensing enzymes from differ- ent Brassica species. Some of these changes indicate that the B. napus cv. Hero allele could be more related to B. oleracea than to the B. rapa FAE1 allele. For example, both Hero and B. oleracea have arginine at position 286, lysine at position 395 and glycine at position 406, while the other FAE1s have glycine, arginine and alanine at these positions respectively. The alignment of our Brassica FAE1 proteins with 3-ketoacyl-CoA synthases from A. thaliana (CUT1 and FAE1) showed that the only highly conserved amino acid residue in all 3-KCSs was the serine 282 residue (Fig. 1). Similar findings were reported by Han et al. [6] when they compared the sequences of FAE1 condensing enzymes from HEA B. napus cv. Ascari and LEA. B. napus cv. Drakkar. Furthermore, in all 3-KCS protein sequences available in the databases, the serine residue at position 282 is conserved or conservatively substituted by threonine (e.g. in Sorghum bicolor, broom corn). Indeed, the single-base change of nucleotide 845 from thymidine to cytosine, which resulted in the substitution of phenylalanine with serine in FAE1 condensing enzyme from LEA cv. Westar at position 282, led for the first time to successful experimental restoration of elongase activity in a previously catalytically inactive enzyme (Fig. 2). The analyses of translation rates of LEA cv. Westar FAE1 condensing enzyme by Western blots showed that translation is not impaired and the strong band corres- ponding to the FAE1 protein of the expected size was detected (Fig. 3). Roscoe et al. [23] reported that the mutations that eliminate 3-KCS activity in LEA rapeseed Fig. 2. GC chromatographs showing fatty acid profiles of transgenic yeast cells. FAMEs were prepared from yeast cell lysates expressing FAE1 condensing enzymes from LEA B. napus cv. Westar wild-type and mutated Westar clones and analyzed by GC. WS-wt, LEA B. napus cv. Westar FAE1, WS-SDM1, site directed mutated clone 1, WS-SDM18, site directed mutated clone 18; pYES2.1, pYES2.1/V5-His-TOPO experimental negative control. Ó FEBS 2002 Restoring enzyme activity in Brassica napus FAE1 (Eur. J. Biochem. 269) 5629 act post-transcriptionally, and that the loss of enzyme activity is related to reduced quantity or stability of the enzyme. Although our results show that the loss of activity is not due to the reduced quantity of protein, our experiments were carried out in a heterologous system. It is possible that the regulation of elongase protein may be quite different in yeast than it is in plants. The immunoblot results clearly indicate that the loss of FAE1 activity is not due to a lower level of expression, since inactive wild-type LEA FAE1 condensing enzyme was expressed in yeast at a level similar to the mutant clones. Recently, Ghanevati & Jaworski [22] studied the role of conserved cysteine and histidine residues in FAE condensing enzyme activity by generating several different mutants, some of them showing complete loss of enzyme activity. Similar to our findings, they concluded that the loss of activity was not related to changes in protein expression level, since their mutant proteins were expressed to the same extent as the FAE1 wild-type protein. It is not unusual that the substitution of a single amino acid results in loss of enzyme activity. Bruner et al.[30] reported that the mutation of aspartate at position 150 to an asparagine residue resulted in nearly complete loss of the peanut oleoyl-PC desaturase (D12 desaturase) activity when the mutated gene was expressed in yeast. Our study constitutes the first mutagenesis of catalytically inactive 3-KCS from a low erucic acid B. napus (canola) cultivar and successful experimental restoration of conden- sing enzyme activity. As mentioned earlier, all our FAE1 expression experi- ments were performed in yeast cells. We expect that the expression of restored FAE1 condensing enzyme from LEA B. napus in planta would result in similar increases in VLCFA content of seed oil as reported by Han et al.[6]. When they expressed FAE1 enzyme from HEA B. napus cv. Ascari in LEA cv. Drakkar, the seed oil of certain transgenic lines contained up to 20% 20:1 and 30% 22:1. They concluded that nonfunctional FAE1 enzyme causes LEA phenotype at the E1 locus. We are now exploring several other FAE1 condensing enzymes from Brassicaceae to enhance our understanding of the role of certain amino acid residues in determining substrate preference and specific activity of these condensing enzymes. ACKNOWLEDGEMENTS We thank Don Schwab, Barry Panchuk and Dr Larry Pelcher of the PBI DNA Technologies Group for primer synthesis and DNA sequencing. We thank Arvind Kumar from Plant Biotechnology Institute (PBI) Seed Oil Modification Group for his technical help with immunoblot preparation, Dr Ljerka Kunst from the University of British Columbia for kindly supplying the anti-FAE1 3-KCS Igs and Dr Jitao Zou from PBI for critical comments and suggestions during the preparation of the manuscript. REFERENCES 1. Somerville, C., Browse, J., Jaworski, J.G. & Ohlrogge, J.B. (2000) Lipids. 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Han, J. & Jaworski, J. (2001) Analysis of isomers of very long chain unsaturated fatty acids in transgenic yeast by GC/MS. Book of Abstracts, p. 29. Biochemistry and Molecular Biology of Plant Fatty Acids and Glycerolipids Symposium, June 6–10, South Lake Tahoe, CA, USA. 6. Han, J., Lu ¨ hs, W., Sonntag, K., Za ¨ hringer, U., Borchardt, D.S., Wolter, F.P., Heinz, E. & Frentzen, M. (2001) Functional char- acterization of b-ketoacyl–CoA synthase genes from Brassica napus L. Plant.Mol.Biol.46, 229–239. Fig. 3. Immunoblot analysis of yeast microsomes expressing FAE1 condensing enzymes. Proteins (100 lg per lane) from yeast expressing wild-type LEA B. napus cv. Westar site-directed mutagenized clones, A. thaliana and empty plasmid pYES2.1 were probed with antibodies raised against the C-terminus of A. thaliana FAE1. Detected protein bands correspond to the FAE1/V5-His fusion protein of approxi- mately 61 kDa (56 kDa FAE1 + 5 kDa V5-His). WS-wt, LEA B. napus cv. 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Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution Vesna Katavic 1 ,. total fatty acids. pYES2 .1/ FAE1 FA (%) 16 :0 16 :1 18:0 18 :1 D9 18 :1 D 11 20 :1 D 11 20 :1 D13 22 :1 D13 22 :1 D15 24 :1 VLCMFA B. n. Hero 16 .34 43.27 3.97 15 .15 1. 52