Báo cáo Y học: Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution pdf

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:
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.
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Ó FEBS 2002 Restoring enzyme activity in Brassica napus FAE1 (Eur. J. Biochem. 269) 5631