Tài liệu Báo cáo Y học: Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases pot

Studies into factors contributing to substrate specificity
of membrane-bound 3-ketoacyl-CoA synthases
Brenda J. Blacklock and Jan G. Jaworski
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA
We are interested in constructing a model for the substrate-
binding site of fatty acid elongase-1 3-ketoacyl CoA synthase
(FAE1 KCS), the enzyme responsible for production of very
long chain fatty acids of plant seed oils. Arabidopsis thaliana
and Brassica napus FAE1 KCS enzymes are highly homo-
logous but the seed oil content of these plants suggests that
their substrate specificities differ with respect to acyl chain
length. We used in vivo and in vitro assays of Saccharomyces
cerevisiae-expressed FAE1 KCSs to demonstrate that the
B. napus FAE1 KCS enzyme favors longer chain acyl sub-
strates than the A. thaliana enzyme. Domains/residues
responsible for substrate specificity were investigated by
determining catalytic activity and substrate specificity of
chimeric enzymes of A. thaliana and B. napus FAE1 KCS.
The N-terminal region, excluding the transmembrane
domain, was shown to be involved in substrate specificity.
One chimeric enzyme that included A. thaliana sequence
from the N terminus to residue 114 and B. napus sequence
from residue 115 to the C terminus had substrate specificity
similar to that of A. thaliana FAE1 KCS. However, a K92R
substitution in thischimeric enzyme changed the specificityto
that of the B. napus enzyme without loss of catalytic activity.
Thus, this study was successful in identifying a domain
involved in determining substrate specificity in FAE1 KCS
andinengineeringanenzymewithnovelactivity.
Keywords: Arabidopsis thaliana; Brassica napus; fatty acid
elongation; 3-ketoacyl-CoA synthase.

The very long chain fatty acids (VLCFA) found in seed
oils are derived from the elongation of products of de novo
fatty acid biosynthesis [1]. The initial reaction of elonga-
tion, i.e. the iterative condensation of acyl units with
malonyl-CoA, is catalyzed in the seed by the membrane-
bound fatty acid elongase-1 3-ketoacyl-CoA synthase
(FAE1 KCS) [2]. Subsequent reduction and dehydration
reactions are carried out by distinct and separate enzymes
that are just beginning to be characterized [1,3,4].
FAE1 KCS was first identified in Arabidopsis thaliana [5]
and homologues have been found in oleaginous species
such as Brassica napus, B. juncea, and Simmondsia chinen-
sis [6–10]. The functional similarity among these enzymes
is demonstrated by the ability of the jojoba FAE1 KCS to
complement the canola fatty acid elongation mutation
even though jojoba produces wax rather than triacylgly-
cerol, as found in other seed oils [6].
Examination of the VLCFA content of the seed oils of
A. thaliana and B. napus reveals differences in the levels of
eicosenoic (20:1) and erucic (22:1) fatty acids. In A. thaliana
seed oil, 20% of the total fatty acids are VLCFA of which
18% of the total fatty acids are in the form of 20:1 and 2%
in the form of 22:1 [11]. In B. napus seed oil, 62% of the
total fatty acids are monounsaturated VLCFA, 10% as 20:1
and 52% as 22:1 [12]. As FAE1 KCS is responsible for
VLCFA production in oilseeds [2,5,6], this diversity in
VLCFA content suggests that A. thaliana and B. napus
FAE1 KCS enzymes have distinct substrate specificities
with the B. napus enzyme favoring longer chain length
substrates than the A. thaliana enzyme. The high sequence

identity (86%) between these two enzymes further suggests
that the determinants responsible for fatty acid substrate
specificity in FAE1 KCS are few and potentially identifi-
able.
The significant amino acid sequence homology of
FAE1 KCSs with soluble condensing enzymes, such as
chalcone synthase and 3-ketoacyl-acyl carrier protein
synthases (KASs) is consistent with a role for FAE1 KCSs
as fatty acid condensing enzymes [5,6,13]. Our understand-
ing of the structure/function relationships of soluble
condensing enzymes has been greatly advanced with the
recent crystal structures of KAS I, -II, and -III and chalcone
synthase [14–19]. However, only limited information is
available about the structure of the membrane-bound
KCSs. Secondary structural analysis of the family of
FAE1 KCS enzymes reveals two putative transmembrane
domains at the N termini of the proteins. Recent work in
our laboratory has confirmed that the amino terminus of
Arabidopsis FAE1 KCS is involved in anchoring the
enzyme to the membrane [20]. The difficulty inherent in
crystallizing membrane-bound enzymes required us to take
a different approach to probing the structure/function
relationships of FAE1 KCS. Here, we report utilization of a
domain-swapping approach to investigate the structural
domains and residues responsible for substrate specificity in
FAE1 KCS.
Correspondence to: B. J. Blacklock, Department of Chemistry and
Biochemistry, Miami University, Oxford, OH 45056, USA.
Fax: +1 513 529 5715, Tel.: +1 513 529 1641,
E-mail:

Abbreviations: VLCFA, very long chain fatty acid; FAE1 KCS, fatty
acid elongase-1 3-ketoacyl CoA synthase; KAS, 3-ketoacyl-acyl
carrier protein synthase; cm-ura, complete minimal dropout media
lacking uracil; FAME, fatty acid methyl ester.
Note: The SWISS-PROT accession numbers for the FAE1 KCS are:
Arabidopsis thaliana, Q38860; Brassica napus, O23738.
(Received 31 May 2002, revised 2 August 2002,
accepted 12 August 2002)
Eur. J. Biochem. 269, 4789–4798 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03176.x
The redesign of a number of plant lipid metabolic
enzymes by swapping domains between related yet func-
tionally divergent enzymes has proven useful in obtaining
catalysts with novel substrate specificity. An understanding
of the regions of enzymes that contribute to substrate
specificity and catalytic activity has also been gleaned from
these studies [21–25]. By replacing only five amino acid
residues, Cahoon and coworkers engineered a soluble fatty
acid desaturase with D
6
-16:0 substrate specificity to one that
functions principally as a D
9
-18:0 desaturase [21]. Similarly,
specificity for a 16:0 substrate was imparted to a D
9
-18:0
desaturase by replacement of a single residue [22].
An analogous approach was taken to deciphering the
architecture of the substrate-binding site of membrane-
bound desaturases and related enzymes [24,26]. Libisch

et al. constructed chimeras of Borago officinalis D
6
-fatty
acid and D
8
-sphingolipid desaturases and analyzed effects
on substrate specificity [24]. These studies were unsuccessful
in identifying a discrete domain that can differentiate
between a phospholipid-conjugated substrate and a cera-
mide-conjugated substrate. However, the authors were
successful in modifying the substrate specificity of the
D
6
-fatty acid desaturase to a preference for shorter chain
fatty acids [24].
A similar study demonstrated a switch in the oxidative
reactions catalyzed by membrane-bound oleate desaturases
and hydroxylases upon site-directed mutagenesis [26]. When
the amino acid sequences of these highly homologous
enzymes were compared, seven residues were identified that
were conserved in the desaturases but divergent in the
hydroxylases. The mutation of these residues in the
desaturases to the corresponding hydroxylase residues
resulted in the conversion of desaturase activity to hydroxy-
lase activity. The reciprocal experiment allowed the conver-
sion of a hydroxylase to a desaturase [26].
The objective of our work was to examine the substrate
specificity of A. thaliana and B. napus FAE1 KCSs and to
study the determinants of fatty acyl chain length specificity
in 3-ketoacyl CoA condensing enzymes. We were able to

map residues and regions of primary structure involved in
substrate specificity in KCS enzymes. These studies repre-
sent the first steps toward a characterization of the substrate-
binding site of the membrane-bound KCS enzymes.
EXPERIMENTAL PROCEDURES
Construction of yeast expression vectors and cell lines
A. thaliana (var. WS) FAE1 and B. napus (var. Golden)
FAE1.1 genes were amplified from plasmids containing the
cDNAs with Vent DNA polymerase (New England
Biolabs) using primers (Table 1) containing restriction sites
convenient for subcloning into the pYES2 expression vector
(Invitrogen). PCR products and DNA fragments were
purified by agarose gel electrophoresis followed by Gene
Clean (Bio101, Vista, CA). A. thaliana, B. napus and
At114K92R genes were subcloned into a pYES2 vector
engineered to encode a (His)
6
GlySer fusion protein through
BamHI/EcoRI restriction enzyme sites. Purified, digested
insert and pYES2 vector were ligated with T4 DNA ligase
(Life Technologies Gibco BRL) and transformed into
competent XL-1 Blue Escherichia coli (Stratagene) by
standard techniques [27]. Insert-containing plasmids were
sequenced by in-house automated DNA sequencing to
ensure they were mutation-free. Plasmids were transformed
into competent Saccharomyces cerevisiae (strain InvSc1,
Invitrogen) by the lithium acetate method of Geitz et al.
[28].
Domain swapping and site-directed mutagenesis
by overlap extension

Chimeric genes were constructed either by restriction
enzyme digestion when convenient sites for domain swap-
ping were present, or by thermostable DNA polymerase-
mediated overlap extension and PCR essentially as
described [29,30]. Briefly, gene fragments were amplified
from plasmids containing A. thaliana or B. napus FAE1
using 5¢ or 3¢ specific primers with restriction sites and
overlapping primers either straddling the desired splice site
of the chimeric gene or containing the desired site-directed
mutation (Table 1). Purified fragments were mixed and the
full-length chimeric gene was constructed by extension of
the overlapping fragments and PCR amplification with
extreme 5¢ and 3¢ primers in the same reaction. Amplifica-
tion products were subcloned into pYES2 and sequenced as
described above to confirm the correct sequence.
Expression of FAE1 KCSs in yeast and preparation
of microsomes
Transformed yeast was grown overnight with shaking in
rich media at 30 °C and was used to inoculate complete
minimal dropout media lacking uracil (cm-ura) [31] supple-
mented with 2% galactose to give an initial D
600
of 0.01–
0.04. The cm-ura + gal cultures were grown to 1.5–2 D
600
units and harvested. Microsomes were prepared as des-
cribed previously [32] using ice-cold isolation buffer (80 m
M
Hepes/KOH pH 7.2, 5 m
M

EGTA, 5 m
M
EDTA, 10 m
M
KCl, 320 m
M
sucrose, 2 m
M
dithiothreitol). Protein con-
centrations were determined after the method of Bradford
[33] using BSA as a standard and adjusted to 2.5 lgÆlL
)1
by
the addition of glycerol to 15% and isolation buffer.
Microsomes were frozen on dry ice, stored at )80 °C until
use and, once thawed, were not refrozen.
GC/MS analysis of yeast lipids
Yeast transformed with pYES2 or pYES2 with FAE1 insert
were grown in cm-ura plus 2% raffinose and were induced
by 2% galactose as described above. Cells were harvested by
centrifugation and washed with dH
2
O; methyl esters of
cellular lipids were prepared by incubation of the cell pellet
in 2% H
2
SO
4
in methanol at 80 °C for 1–2 h. Fatty acid
methyl esters (FAMEs) were extracted into hexanes,

concentrated by evaporation under an N
2
stream and
dissolved in a small volume of hexanes. FAMEs were
separated by GC (Thermoquest Trace GC) on an RTX-
5MS 0.25 lm column (Restek Corp. Bellefonte, PA) and
identifiedbyMSonaninlineFinniganPolarismass
spectrometer.
Assay of elongation activity of FAE1 KCSs
Fatty acid elongase activity was measured essentially as
described previously [34]. The elongation reaction consisted
4790 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002
of 20 m
M
Hepes/KOH pH 7.2, 20 m
M
MgCl
2
, 500 l
M
NADPH, 10 l
M
CoASH, 100 l
M
malonylCoA, 15 l
M
[1-
14
C]18:1CoA, and 6 lg protein of prepared microsomes
in a final volume of 25 lL. [1-

14
C]18:1CoA was synthesized
from [1-
14
C]18:1 free fatty acid (50–55 lCiÆlmol
)1
;ICN,
Costa Mesa CA) as described by Taylor et al.[35].Methyl
esters of the radiolabeled acyl CoA elongation products
were prepared as described above, separated by reversed
phase silica gel TLC (Alltech, Deerfield, IL) with acetonit-
rile/methanol/H
2
O (65 : 35 : 0.5, v/v) developing solvent
[36] and analyzed by phosphorimaging; each band was then
quantitated by ImageQuant software (Molecular Dynam-
ics, Inc.).
Solubilization and isolation of (His)
6
-tagged fusion
proteins
(His)
6
fusion proteins were solubilized and isolated as
described [20]. Microsomes (2 lgproteinÆlL
)1
) expressing
(His)
6
-tagged fusion proteins were solubilized by incubation

on ice for 2 h in solubilization buffer (320 m
M
NaCl, 0.5%
Triton X-100) and insoluble material was pelleted by
ultracentrifugation at 4 °C for 1 h at 235 000 g. Superna-
tants were combined with binding buffer (25 m
M
sodium
phosphate pH 8.0, 0.5% Triton X-100, 150 m
M
NaCl, 10%
glycerol) at a ratio of 0.43 : 1 (supernatant : binding buffer)
and applied to a 300-lL column of Ni
+2
-pentadentate
chelator (Ni-PDC, Affiland, Ans-Liege, Belgium). The
column was washed with 1 mL binding buffer, 1 mL wash
buffer (25 m
M
sodium phosphate pH 8.0, 0.5% Triton
X-100, 500 m
M
NaCl, 10% glycerol, 20 m
M
imidazole) and
1 mL binding buffer. Proteins were eluted from the column
with 300 lL elution buffer (25 m
M
sodium phosphate
pH 8.0, 0.5% Triton X-100, 150 m

M
NaCl, 10% glycerol,
300 m
M
imidazole), dithiothreitol was added to 2 m
M
, and
samples were frozen on dry ice and stored at )80 °C.
Condensation assay of (His)
6
-FAE1 KCS
Condensation activity of (His)
6
-FAE1 KCSs was assayed as
previously described [37]. Briefly, 2.5 lL purified sample
was incubated with condensation reaction mix (10 m
M
sodium phosphate, pH 7.2, 0.05% Triton X-100, 15 l
M
acyl CoA, 20 l
M
[3-
14
C]malonyl CoA) where the acyl CoA
Table 1. Primers used in subcloning and overlap PCR.
Primer Name Sequence
A. thal.5¢ BamHI 5¢-GGGGATCCATGACGTCCGTTAACGTTA3¢
A. thal.3¢ EcoRI 5¢-CCCGAATTCTTAGGACCGACCGTTTTGGACATGAGTCTT-3¢
B. nap.5¢ BamHI 5¢-GGGGATCCATGACGTCCATTAACGTAAAGCTCC-3¢
B. nap.3¢ EcoRI 5¢-CCGAATTCTTAGGACCGACCGTTTTGG-3¢

At173sense 5¢-GCGCTCGAAAATCTATTCAAGAACACC-3¢
At173anti 5¢-GTTCTTGAATAGATTTTCGAGCGCACCGATGATAAC-3¢
Bn173sense 5¢-GCCCTAGAAAATCTATTCAAGAACACC-3¢
Bn173anti 5¢-GTTCTTGAATAGATTTTCTAGCGCACCATT-3¢
At399sense 5¢-GCCGGAGGCAGAGCCGTGATCGAT-3¢
At399anti 5¢-ATCGATCACGGCTCTGCCTCCGGC-3¢
Bn399sense 5¢-GCCGGAGGCAGAGCCGTGATCGAT-3¢
Bn399anti 5¢-ATCGATCACGGCTCTGCCTCCGGC-3¢
At74sense 5¢-CCCAAACCGGTTTACCTCGTTGA-3¢
At74anti 5¢-TCAACGAGGTAAACCGGATTGGG-3¢
At114sense 5¢-CGGAACGGCACGTGTGATGATTCGTCCT-3¢
At114anti 5¢-GGACGGATCATCACACGCGACGTTCCG-3¢
At114D81Eanti 5¢-GTAACACG GTACTCAACGAGATAAAC-3¢
At114D81Esense 5¢-GTTTATCTCGTTGAGTACTCGTGTTAC3¢
At114P89Tanti 5¢-AACTTTGAGATGCGTTGGCGGAAGGTA-3¢
At114P89Tsense 5¢-TACCTTCCGCCAACGCATCTCAAAGTT-3ı
´
At114L91Canti 5¢-GACACTAACTTTACAATGCGTGGCGG3¢
At114L91Csense 5¢-CCGCCACCGCATTGTAAAGTTAG GTC-3¢
At114K92Ranti 5¢-AGAGACACTAACTCTGAGATGCGGTGG-3¢
At114K92Rsense 5¢-CCACCGCATCTCAGAGTTAGTGTCTCT-3¢
At114V93Santi 5¢-TTTAGAGACACTTGATTTGAGATGCGG3¢
At114V93sense 5¢-CCGCATCTCAAATCAAGT GTCTCTAAA-3¢
At114V95Ianti 5¢-CATGACTTTAGAGATACTAACTTTGAG-3¢
At114V95Isense 5¢-CTCAAAGTTAGTATCTCTAAAGTCATG-3¢
At114I105Vanti 5¢-ATCAGCTTTTCTTACTTGGAGAAAAT-3¢
At114I105Vsense 5¢-ATTTTCTACCAAGTAAGAAAAGCTGAT-3¢
At114T110Panti 5¢-GTTCCGTGAAGAAGGATCAGCTTTTCT-3ı
´
At114T110Psense 5¢-AGAAAAGCTGACCTTCTTCACGGAAC-3¢

At114S112-anti 5¢-CGTGCCGTTCCGAGAAGTATCAGC-3¢
At114S112-sense 5¢-GCTGATACTTCTCGGAACGGCACG-3¢
AtK92R sense 5¢-CCACCGCATCTCAGAGTTAGTGTCTCT-3¢
AtK92R anti 5¢-AGAGACACTAACTCTGAGATGCGGTGG-3¢
Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4791
was either 18:1CoA or 20:1CoA for 10 min at 30 °C.
Radiolabeled malonyl CoA was prepared as described
previously [38]. 3-ketoacyl CoA reaction products were
reduced to the diols with NaBH
4
, extracted into petroleum
ether, concentrated by evaporation under an N
2
stream, and
quantified by liquid scintillation analysis.
RESULTS
This report describes studies of the fatty acid chain length
substrate specificity of FAE1 KCS. We tested the hypothe-
sis that A. thaliana and B. napus FAE1 KCS enzymes have
divergent substrate specificity and that determinants of that
specificity can be identified by domain swapping experi-
ments.
We have developed a facile biochemical method to
characterize fatty acid elongase activities using an S. cere-
visiae expression system. Endogenous yeast fatty acids serve
as elongation substrates for in vivo analysis [2] and
exogenously supplied radiolabeled 18:1CoA is readily taken
up by microsomal fractions for in vitro assays [39].
As a first step, we established that the A. thaliana and
B. napus FAE1 KCSs were active in the S. cerevisiae

expression system and indeed had distinct substrate speci-
ficities. Elongation of yeast endogenous fatty acids by
FAE1 KCSs was examined by expressing plant
FAE1 KCSs in yeast and then analyzing the fatty acid
content of cellular lipids. Table 2 shows the C20 to C26
fatty acid content of cellular lipids when yeast transformed
with pYES2 with A. thaliana or B. napus FAE1 inserts or
the empty vector was grown under galactose induction
conditions. The VLCFA content of yeast carrying the
empty vector was principally limited to 26:0 with little or no
C20, C22 or C24 fatty acids present. In contrast, expression
of either A. thaliana or B. napus FAE1 KCS resulted in the
production of significant levels of both saturated and
unsaturated C20 to C26 VLCFA. A. thaliana FAE1 KCS
expression caused an increase in VLCFA methyl esters to
32% of the total FAMEs: 19.3% C20, 5.8% C22, 2.6% C24
and 4.3% C26. B. napus FAE1 KCS expression, on the
other hand, produced 11% of the total FAMEs as VLCFA:
3.5% C20, 2.1% C22, 0.8% C24 and 4.1% C26. These data
demonstrate that plant FAE1 KCS enzymes were expressed
and have activity in this yeast system. Further, these results
show that in yeast, A. thaliana FAE1 KCS produces more
C20, C22 and C24 fatty acids than B. napus FAE1 KCS
and that the majority of the VLCFA products are C20.
B. napus FAE1 KCS appeared to have more activity
toward C20 than A. thaliana FAE1 KCS as indicated by
near equal levels of C20 and C22 products when B. napus
FAE1 KCS was expressed. While these results are, in
general, consistent with the apparent substrate specificity of
the A. thaliana and B. napus FAE1 KCS enzymes in seeds,

the B. napus enzyme appeared to be less active than the
A. thaliana FAE1 KCS in yeast.
The substantial levels of saturated and unsaturated fatty
acid methyl esters demonstrated that FAE1 KCSs have
activity toward both monounsaturated and saturated fatty
acids. The FAE1 KCS-derived saturated fatty acid prod-
ucts appear to feed into the yeast saturated VLCFA
pathway. VLCFA up to C26 as 24:0 and 26:0 were
produced at elevated levels when either A. thaliana or
B. napus FAE1 KCS were expressed compared to the
empty vector control.
While in vivo assays indicated that the plant FAE1 KCSs
were able to couple to the yeast fatty acid elongation
complex, an assay of the actual catalytic activity of the
FAE1 KCSs was required to characterize the enzymes
accurately. Microsomes were prepared from galactose-
induced yeast transformed with empty vector (pYES2) or
pYES2 with A. thaliana or B. napus FAE1 insert. Elonga-
tion of [1-
14
C]18:1CoA to [3-
14
C]20:1CoA and
[5-
14
C]22:1CoA by the microsomes was measured by
incubation with malonyl CoA and required cofactors,
followed by transacylation of the CoA conjugates to methyl
esters [34,39]. Elongation activity of both A. thaliana and
B. napus FAE1 KCS reached a plateau by 20 min but the

catalytic activity of B. napus FAE1 KCS was lower than
that of A. thaliana FAE1 KCS (Fig. 1). When the activities
of the two enzymes were compared in numerous experi-
ments with individual transformants, this difference
between A. thaliana and B. napus FAE1 KCSs was consis-
tently observed. As the same expression vector and yeast
strain were used, this appears to reflect actual differences in
catalytic activity rather than differences in expression level
although we do not know the amount of FAE1 KCS
protein present in the microsomes. It appears that the
A. thaliana FAE1 allele codes for a more active enzyme than
the B. napus FAE1 allele used here. Subsequently, we were
able to isolate (His)
6
-tagged fusion proteins and directly
assay condensation activity of A. thaliana and B. napus
(His)
6
-FAE1 KCSs [A. thaliana, 538.5 ± 68.1 pmolÆlg
)1
protein (± SD) 20:1 productand 126.8 ± 14.7 22:1 product;
B. napus 135.0 ± 17.5 20:1 product and 64 ± 4.4 22:1
product]. These observations are consistent with results
obtained in in vivo experiments.
Comparison of total elongation activities demonstrated
that the A. thaliana enzyme was more active than the
Table 2. Effect of the expression of A. thaliana and B. napus FAE1 KCS on yeast cellular lipids. S. cerevisiae transformed with expression vectors
encoding A. thaliana or B. napus FAE1 KCS or the empty vector (pYES2) were pre-grown on raffinose (2%) and induced with galactose (2%) for
2 days. Total fatty acid composition was determined by GC/MS of FAMEs prepared by transacylation. Results are reported as the percentage total
fatty acid methyl esters and are the mean ± SD of five independent transformants.

FAME (%)
20:1 20:0 22:1 22:0 24:1 24:0 26:0
pYES2 0.17 ± 0.03 0.23 ± 0.05 0.07 ± 0.01 0.00 ± 0.01 0.00 ± 0.00 0.06 ± 0.01 1.57 ± 0.33
A. thaliana 17.29 ± 2.52 2.01 ± 0.46 4.32 ± 0.67 1.46 ± 0.29 0.60 ± 0.16 1.99 ± 0.39 4.29 ± 1.30
B. napus 2.47 ± 0.6 1.02 ± 0.24 1.61 ± 1.06 0.50 ± 0.22 0.19 ± 0.08 0.59 ± 0.19 4.13 ± 1.26
4792 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002
B. napus enzyme (Table 3). Analysis of the individual acyl
products indicated differences in the substrate specificity
of the two FAE1 KCSs. Relative substrate specificity of
FAE1 KCSs can be compared by expressing the ratio of
22:1 produced over 20:1 produced. An enzyme which favors
the production of 22:1 would therefore have a higher 22:1/
20:1 product ratio than one which favors the production of
20:1 fatty acids. Table 3 shows the product ratios of
A. thaliana and B. napus FAE1 KCSs after 10 min The
larger 22:1/20:1 product ratio of B. napus FAE1 KCS
demonstrated that B. napus FAE1 KCS was indeed pro-
portionally more active toward 20:1 as a substrate than the
A. thaliana FAE1 KCS. A. thaliana FAE1 KCS had a
substrate specificity directed primarily toward an 18:1
substrate.
Both in vivo and in vitro data indicate that A. thaliana
and B. napus FAE1 KCS enzymes have distinct substrate
specificities and that differences in seed oil composition
reflect these substrate specificities. The primary sequence
alignment (Fig. 2) demonstrates that there is a high degree
of homology between A. thaliana and B. napus
FAE1 KCS (86% identity). This similarity suggested to
us that factors involved in substrate specificity were
potentially identifiable. As little is known about the

structure of FAE1 KCSs, we approached the problem
with no attempt to predict where residues or regions
important in substrate specificity would be found. Swap-
ping of domains between A. thaliana and B. napus
FAE1 KCSs by ligation of restriction digest fragments or
by overlap PCR produced cognate full-length chimeric
FAE1 genes. Fig. 3 is a schematic representation of a
number of the chimeric enzymes engineered in this study.
Chimeric enzymes were characterized based on activity and
the 22:1/20:1 product ratio. Our goal was to identify the
smallest domain required to retain a particular substrate
specificity and to engineer an enzyme with novel activity.
Twenty-nine chimeras were prepared for this study.
The first group of constructs produced pairs of chimeras
with switchover points between A. thaliana FAE1 KCS
sequence and B. napus FAE1 KCS sequence at residues
399, 254 and 173. These constructs represent swaps in the
C-terminal domain, at the midpoint and in the N-terminal
domain (Fig. 3). Chimeras were named based on the origin
of the N terminus and the residue at which the switchover
occurred. For example, for At254, residues 1–254 are
derived from A. thaliana FAE1 KCS and residues 255–506
are derived from B. napus FAE1 KCS. When in vitro
elongation activity was measured, the initial FAE1 KCS
chimeras segregated into two classes: those with measurable
elongase activity and those with little or no activity
(Fig. 4A). All of the chimeric enzymes with activity had
A. thaliana sequence at the N terminus while enzymes with
B. napus coding sequence in the N terminus had little or no
activity. All of the active chimeras had A. thaliana-like

substrate specificity as indicated by low 22:1/20:1 product
ratios (Fig. 4B). The FAE1 KCS chimera with the smallest
region containing A. thaliana FAE1 KCS sequence in this
group was At173 indicating that at least one determinant of
Table 3. Elongation activity and product ratio (22:1/20:1) for A. thaliana and B. napus FAE1 KCS. Reactions were carried out for 10 min and
results presented are the mean ± SD of five individual assays using four separate microsomal preparations.
Elongation activity (pmolÆlg
)1
protein)
20:1 + 22:1 20:1 22:1 22:1/20:1
A. thaliana 12.04 ± 1.80 10.72 ± 0.60 1.32 ± 0.36 0.12 ± 0.01
B. napus 7.39 ± 0.60 5.52 ± 1.2 1.87 ± 0.90 0.34 ± 0.1
16
14
12
10
8
6
4
2
0
elongation activity (pmol/
µ
g protein)
50403020100
time (min)
Fig. 1. Elongase activity of microsomes prepared from S. cerevisiae
expressing A. thal iana or B. napus FAE1 KCS. Microsomes prepared
from induced S. cerevisiae transformed with empty vector (pYES2, n)
or vectors encoding A. thaliana (h)orB. napus (s)FAE1 KCSswere

assayed for the conversion of [1-
14
C]18:1CoA to [3-
14
C]20:1CoA and
[5-
14
C]22:1CoA as described in Experimental procedures. Time points
were taken between 5 and 45 min and are plotted as total elongation
products (20:1 + 22:1, pmolÆlg
)1
protein) vs. time. The presented
results are the mean of five assays using four separate microsomal
preparations and error bars indicate standard deviation.
Fig. 2. Alignment of A. thaliana (top) and B. napus (bottom)
FAE1 KCS sequences. Identical residues are included in the shaded
box.
Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4793
fatty acyl chain length specificity in FAE1 KCSs resides in
the N terminal one-third of the protein.
We further dissected the N-terminal region of the
FAE1 KCSs by preparing chimeras with switchover
points at residues 114 and 74 (At114 and At74). Although
the catalytic activity was lower in these chimeras, At114
had A. thaliana FAE1 KCS-like substrate specificity while
At74 more closely resembled B. napus FAE1 KCS in
substrate specificity (Table 4). At74 had primarily
B. napus sequence (85%) with A. thaliana sequence inclu-
ded only in the extreme N-terminal putative transmem-
brane domains. Thus, a shift in substrate specificity from

A. thaliana FAE1 KCS-like to B. napus FAE1 KCS-like
occurred between chimera At114 and At74 indicating that
determinants of substrate specificity reside between resi-
dues 74 and 114. Furthermore, as At74 is encoded entirely
by B. napus sequence except for the transmembrane
domain, the transmembrane domain appears to have
little or no role in the determination of substrate
specificity in FAE1 KCS.
The stretch of primary sequence between residues 74 and
114 contains nine nonidentical residues (Fig. 2). Site-direc-
ted mutagenesis of chimera At114 was utilized to dissect this
region further in an attempt to reveal specific residues
involved in imparting specificity toward the 20:1 substrate in
FAE1 KCSs. Some of these chimeras were inactive
(At114D81E, At114P89T, At114L91C, At114V93S), while
others had activity similar to that of the parent enzymes
(Fig. 5A). When the 22:1/20:1 product ratio of the active
chimeras were compared, all chimeras in this group had
A. thaliana-like product ratios except At114K92R, which
had the substrate specificity of B. napus FAE1 KCS
(Fig. 5B). This suggests that residue 92 has some role in
determining substrate specificity of FAE1 KCSs. Catalytic
activity of At114K92R, however, was similar to that of
A. thaliana FAE1 KCS (Fig. 5A) while At114 had lower
catalytic activity than A. thaliana FAE1 KCS. This dem-
onstrated a novel activity in At114K92R and suggested that
residue 92 is involved in catalytic activity as well as substrate
specificity.
We further examined the role that residue 92 plays in
substrate specificity by replacing K92 with R in wild-type

A. thaliana FAE1 KCS. Both catalytic activity and
25
20
15
10
5
0
elongation activity (pmol/
µ
g protein)
pYES2
A. thaliana
B. napus
At399
Bn399
At254
Bn254
At173
Bn173
A
0.6
0.5
0.4
0.3
0.2
0.1
0.0
product ratio 22:1/20:1
A. thaliana
B. napus

At399
At254
At173
B
Fig. 4. (A) Elongation activity (20:1 + 22:1, pmolÆlg
)1
protein) for
A. thaliana, B. napus, At399, Bn399, At254, Bn254, At173, and Bn173
FAE1 KCS and (B) product ratio (22:1/20:1) for A. thaliana, B. napus,
At399, At254, and At173 FAE1 KCS. (A) Reactions were carried out
for 10 min and results presented are the mean of one to three indi-
vidual assays using three microsomal preparations (± SD). (B)
Reactions were carried out for 10 min and results presented are
the mean of one to three individual assays using three microsomal
preparations (± SD).
Fig. 3. Schematic representation of chimeric FAE1 KCS polypeptides.
A. thaliana FAE1 KCS sequence is represented as an open bar
and B. napus FAE1 KCS sequence is represented as a shaded bar.
The chimeric alleles were named for the FAE1 KCS sequence in
the N-terminal domain followed by the residue number at which the
sequence shifts to the other FAE1 KCS sequence. Point mutations are
named by the convention of the wild-type residue followed by the
residue number and the amino acid that has been substituted at that
residue.
Table 4. Elongation activity and product ratio (22:1/20:1) for A. t hali-
ana, B. napus,At114andAt74FAE1KCS.Reactions were carried out
for 10 min and results presented are the mean ± SD of two or three
individual assays for each of four microsomal preparations.
Elongation activity (pmolÆlg
)1

protein)
20:1 + 22:1 22:1/20:1
A. thaliana 16.7 ± 2.4 0.13 ± 0.01
B. napus 11.9 ± 3.6 0.33 ± 0.10
At74 7.2 ± 2.3 0.36 ± 0.06
At114 10.0 ± 2.7 0.15 ± 0.03
4794 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002
substrate specificity of AtK92R were essentially identical to
the wild-type A. thaliana FAE1 KCS (Fig. 5).
A procedure for the isolation of (His)
6
-tagged
FAE1 KCSs was developed in our laboratory, subsequent
to the analysis of the entire set of chimeras prepared in this
study, which allowed us to assay directly the condensation
activity of the isolated (His)
6
-FAE1 KCSs [20]. We used this
assay to verify that the product ratios observed in the
elongation assay of microsomal preparations were an actual
measure of the activity of the FAE1 KCS and were not an
artifact of differential interactions with yeast enzymes
required to produce the elongation product. When the
condensation activity of isolated (His)
6
-tagged A. thaliana,
B. napus, and At114K92R FAE1 KCSs were measured
with 18:1CoA and 20:1CoA substrates, 22:1/20:1 product
ratios were: (His)
6

-A. thaliana FAE1 KCS, 0.25 ± 0.07;
(His)
6
-B. napus FAE1 KCS, 0.78 ± 0.13; (His)
6
-
At114K92R, 0.71 ± 0.10 (product ratio ± SD, n ¼ 9).
The product ratio of condensation activity of (His)
6
-
B. napus FAE1 KCS and (His)
6
-At114K92R was therefore
similar and distinct from that of (His)
6
-A. thaliana
FAE1 KCS as was demonstrated by the elongation assay
of FAE1 KCSs in the yeast microsomal system.
DISCUSSION
In this study, we attempted to gain insights into the
structural basis for substrate specificity in FAE1 KCS
enzymes. As no crystal structure of any membrane-
bound FAE1 KCS is available, we used a biochemical
approach with molecular genetic tools. We reasoned that
the difference in oil content of A. thaliana and B. napus
seeds is a consequence of divergent substrate specificity of
the key enzyme responsible for VLCFA production in
seeds, FAE1 KCS. The high degree of homology between
these enzymes allowed us to use a domain-swapping
approach to identify regions/residues involved in substrate

specificity.
Our first step was to establish the substrate specificity of
A. thaliana and B. napus FAE1 KCSs with both in vivo and
in vitro assays of elongation activity. Analysis of the effect of
expression of A. thaliana and B. napus FAE1 KCS on yeast
cellular lipids indicated that these enzymes have activity
toward both monounsaturated and saturated fatty acids.
This is consistent with the observation by Millar and Kunst
that over-expression of FAE1 KCS in Arabidopsis resulted
in an increase in elongation products of both 18:0 and 18:1
fatty acids [2]. In addition, James et al. showed that
disruption of expression of FAE1 KCS in Arabidopsis
resulted in a decrease in the levels of both saturated and
monounsaturated VLCFA in seed [5]. In the in vivo
experiment presented here, C18 and C20 monounsaturated
fatty acids were elongated by the FAE1 KCSs to a greater
degree than were saturated fatty acids. This may reflect the
available substrates in this yeast system and/or the ability of
the enzymes further down the metabolic pathway to use the
products of the FAE1 KCS condensation activity. Indeed,
in the S. cerevisiae strain used in these studies, monoun-
saturated C16 and C18 fatty acids were at least twice as
abundant as the saturated C16 and C18 fatty acids.
The in vivo activities presented here demonstrated that
both FAE1 KCS enzymes were active in the yeast expres-
sion system and were able to couple to the yeast fatty acid
elongation complex. These results show the usefulness of
this in vivo system as a quick screen for activities of
FAE1 KCSs. However, the results are reflective of the entire
yeast metabolic pathways from the synthesis of 3-ketoacyl

CoAs by a condensing enzyme to the incorporation of fatty
acids into lipids such as triacylglycerol or membrane
phospholipids. This in vivo system is therefore inadequate
for assigning substrate specificity and catalytic activity to an
exogenous KCS as activities and specificities of subsequent
yeast enzymes, potential compartmentalization and avail-
ability of substrates could interfere with interpretation of the
results. This may be especially important in examining the
activity of other plant KCS enzymes such as those encoded
by KCS1 and CER6 which use longer VLCFA as substrates
[39,40]. A detailed examination of activity and substrate
specificity of a KCS is possible only with an in vitro
approach.
We found that in both in vivo and in vitro assays,
A. thaliana FAE1 KCS had more activity toward a C18
substrate than toward a C20 substrate. The B. napus
enzyme, on the other hand, had similar activity toward
C18 and C20 fatty acyl groups. The data presented
here demonstrate that A. thaliana and B. napus
FAE1 KCS enzymes have distinct substrate specificities
20
15
10
5
0
elongation products (pmol/
µ
g protein)
A. thaliana
B. napus

At114D81E
At114P89T
At114L91C
At114K92R
At114V93S
At114V95I
At114I105V
At114T110P
At114S112-
AtK92R
0.5
0.4
0.3
0.2
0.1
0.0
product ratio 22:1/20:1
A. thaliana
B. napus
At114K92R
At114V95I
At114I105V
At114T110P
At114S112-
AtK92R
B
A
Fig. 5. (A) Elongation activity (20:1 + 22:1, pmolÆlg
)1
protein) for

A. thaliana, B. na pus, At114D81E, At114P89T, At114L91C,
At114K92R, At114V93S, At114V95I, At114I105 V, At114T110P,
At114S112-, AtK92R FAE1 KCS and (B) product ratio (22:1/20:1) for
A. thaliana, B. napus, At114K92R, At114V95I, At114I105 V,
At114T110P, At114S112-, AtK92R FAE1 KCS. (A) Reactions were
carried out for 10 min and results presented are the mean of one to
three individual assays each of at least seven separate microsomal
preparations (± SD). (B) Reactions were carried out for 10 min and
results presented are the mean of one to three individual assays each of
at least seven separate microsomal preparations (± SD).
Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4795
and are consistent with our hypothesis that differences in
seed oil composition reflect these substrate specificities.
Through the preparation and assay of chimeric enzymes,
we were able to define the region between residue 74 and 173
as an important domain in conferring substrate specificity to
FAE1 KCSs. This domain excludes the putative transmem-
brane domain predicted to be to the N-terminal side of
residue 72. It is not surprising that the transmembrane
domain of FAE1 KCSs is not involved in substrate
specificity and therefore substrate binding because the fatty
acyl CoA substrates are expected to be delivered from the
cytosol and available at the membrane interface [1]. This is
in contrast to membrane-bound fatty acid- and sphingo-
lipid-desaturases, which appear to utilize phospholipid- or
sphingolipid-conjugated fatty acids. Chimerigenesis studies
of the borage D
6
-fatty acid and D
8

-sphingolipid desaturases
indicate that a transmembrane portion of these enzymes is
involved in substrate binding which is consistent with the
membrane localization of the substrates of these desaturases
[24].
If the region from the end of the transmembrane domain
to residue 173 is indeed involved in imparting substrate
specificity to KCS condensing enzymes, we would expect
this region to be of particular heterogeneity among the
family of plant ketoacyl-CoA condensing enzymes. The
completion of the sequence of the Arabidopsis genome [41]
presents an opportunity to compare the sequences of all
predicted KCS-like genes in the genome. A recent database
search revealed at least 15 distinct KCS-like genes in the
Arabidopsis genome. The gene product of four of these have
been assigned physiological functions in mutant analysis
and expression disruption studies; FAE1, CER6, KCS1,
and FDH [2,5,39,40,42,43]. Alignment of these sequences
demonstrated regions of high homology distributed
throughout the enzymes. The region corresponding to
residues 74–173 of A. thaliana FAE1 KCS, however, is
relatively heterogeneous. Recent work in our laboratory has
identified the active site cysteine of A. thaliana FAE1 KCS
as residue 223 and has suggested that His391 is also involved
in the active site [44]. These two catalytically important
residues are found in regions of high homology and are
distant from the region that has been identified to be
important in substrate specificity in this study.
Given the similarity of the two interchanged residues, it is
surprising that the change of K92 to R in At114 resulted in a

shift of substrate specificity from A. thaliana-like to one
more specific for a 20:1 substrate. When the substitution of
amino acids in families of proteins such as globins and
cytochrome c was calculated, arginine and lysine were often
substituted for each other and were considered ÔsafeÕ
substitutions [45]. The absence of an effect of the K92R
mutation in wild-type A. thaliana FAE1 KCS points to a
role for residue 92 in determining substrate specificity that is
specific to At114 FAE1 KCS. Arg92 in At114K92R
appears to interact with B. napus residues that are not
present in A. thaliana FAE1 KCS. These results also
indicate that residue 92 is not the sole residue involved in
conferring substrate specificity in FAE1 KCS. The chimera
At114K92R will therefore be invaluable in further studies of
the domains/residues that are involved in substrate speci-
ficity in FAE1 KCS as this enzyme may now be utilized in
domain-swapping experiments that focus on the C termini
of the proteins.
We noted the levels of enzyme activity of A. thaliana and
B. napus FAE1 KCSs with interest. B. napus seed oil has a
larger proportion of VLCFA compared to total fatty acids
than A. thaliana seed oil. The incongruity of the level of seed
oil VLCFA and the activity of the B. napus FAE1 KCS
assayed here may have several explanations. A less active
FAE1 KCS in B. napus seeds could be compensated by the
efficiency of the remainder of the biosynthetic pathway. At
least five very similar genes encoding FAE1 KCS enzymes
have been found in B. napus cultivars [7,8,46,47]. The fatty
acid content of the seed oils of the cultivars from which these
genes were cloned may reflect differences in elongation

activity. The FAE1 KCS from B. napus (var. Golden) used
in this study may have a relatively low level of activity
compared to other B. napus FAE1 KCS homologues. In
addition, the amphidiploidy of Brassica species such as
B. napus [12] may result in a higher seed oil VLCFA level
than that predicted from the in vitro activities of individual
FAE1 KCSs. In the plant, one highly active FAE1 KCS
may dominate or the additive effect of expression of
two different FAE1 KCSs may result in high VLCFA
production.
In this work we also noted the level of activity found in
many of the chimeric enzymes. Many of the changes we
made resulted in chimeric enzymes with substantially
lower activity than the wild-type parents. This was
especially apparent when the N terminus of the chimeras
was derived from B. napus FAE1 KCS. In addition,
chimeras At74 and At114 had relatively poor activity
compared to the wild-type enzymes. When similar experi-
ments were conducted with plant acyl-acyl carrier protein
thioesterases and acyl-acyl carrier protein desaturases, a
decrease in the activity of the engineered enzymes often
resulted, regardless of a high level of identity between the
two enzymes examined [21–23]. On the other hand,
several of the changes made in our study resulted in
enzymes with excellent retention of activity. For example,
At114K92R had even more activity than the At114 parent
chimera.
The crystal structures of related condensing enzymes,
E. coli KAS I, -II, and -III, alfalfa chalcone synthase and
S. cerevisiae 3-ketoacyl-CoA thiolase, have been solved [15–

19,48]. Although the overall sequence homology is very low,
these enzymes exhibit a common fold with a five-layered
core structure; a-b-a-b-a, where a comprises two a-helices
and each b is a five-stranded, mixed b-sheet [16,18,19].
Alignment of the secondary structural features of the crystal
structures with
PHD
predicted secondary structure of
FAE1 KCS (data not shown) suggesting that there are
common structural elements among these condensing
enzymes. The substrate-binding site of KAS II may repre-
sent the best available model for that of FAE1 KCS, as
KAS II catalyzes the condensation of a 16:1 moiety with
malonyl-ACP. The crystal structure of KAS II alone and
with the mycotoxin inhibitor, cerulenin, revealed that the
substrate binding pocket of KAS II is lined with hydro-
phobic residues predominantly from the N-terminal domain
of the enzyme that are essential for the binding of long chain
substrates such as 16:1 [16,49]. The research presented here
demonstrates that residues important to substrate binding
for FAE1 KCS also include N-terminal residues and may
suggest similar substrate-binding pockets for the two
enymes.
4796 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002
In the redesign of a soluble fatty acid desaturase from one
with D
6
-16:0 activity to one with D
9
-18:0 activity, Cahoon

et al. replaced only five amino acids in the D
6
-16:0 specific
enzyme with corresponding residues from the D
9
-18:0
specific desaturase [21]. The X-ray crystal structure of the
D
9
-18:0 desaturase revealed that many of the residues which
were identified in the mutagenesis study to be responsible
for chain length substrate specificity, line the substrate-
binding pocket [21,50]. The substitution of only two residues
is sufficient for the conversion of an 18:0-specific desaturase
to one that strongly prefers a 16:0 substrate albeit with less
than half of the total catalytic activity of the parent enzyme
[21]. Taken together with our studies, these results suggest
that the substrate-binding site of the soluble desaturases is
more rigid in the nature of acceptable substrates than that of
FAE1 KCS. The KCS enzymes studied here appear to have
a flexible substrate-binding pocket as demonstrated by the
in vivo activities in yeast. The conversion of an 18:1-specific
condensing enzyme to one that uses only 20:1 substrates
may not therefore be feasible with the replacement of a
small number of residues.
In summary, this work was successful in identifying, for
the first time, regions and residues important in fatty acyl
chain length specificity in a membrane-bound condensing
enzyme. The identification of At114K92R as an enzyme
with novel activity will be useful for the future identification

of other residues involved in fatty acyl chain length substrate
specificity in FAE1 KCS. This study will also serve as a basis
for progress toward further understanding the structure–
function relationships for FAE1 KCS and other membrane-
bound fatty acid elongase condensing enzymes.
ACKNOWLEDGEMENTS
We thank Dr Hugo Dooner, Rutgers University, for permission to use
the A. thaliana FAE1 gene and Dr Ljerka Kunst, University of British
Columbia, for the generous gift of the B. napus FAE1 cDNA. This
work was supported by a grant from Cargill, Inc.
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