The variant ‘his-box’ of the C18-v9-PUFA-speci¢c elongase IgASE1
from Isochrysis galbana is essential for optimum enzyme activity
Baoxiu Qi?, Thomas C.M. Fraser, Claire L. Bleakley, Elisabeth M. Shaw, A. Keith Stobart,
Colin M. Lazarus
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK
Received 15 April 2003; accepted 19 May 2003
First published online 18 June 2003
Edited by Richard Cogdell
ic fatty acid elongase component from Isochrysis galbana, con-
tains a variant histidine box (his-box) with glutamine replacing
the ¢rst histidine of the conserved histidine-rich motif present in
all other known equivalent proteins. The importance of gluta-
mine and other variant amino acid residues in the his-box of
IgASE1 was determined by site-directed mutagenesis. Results
showed that all the variation in amino acid sequence between
this motif in IgASE1 and the consensus sequences of other
elongase components was required for optimum enzyme activity.
The substrate speci¢city was shown to be una¡ected by these
changes suggesting that components of the his-box are not di-
rectly responsible for substrate speci¢city.
? 2003 Federation of European Biochemical Societies. Pub-
lished by Elsevier Science B.V. All rights reserved.
IgASE1, a C18-v v9-polyunsaturated fatty acid-specif-
Key words: Fatty acid elongating activity;
Site-directed mutagenesis; Speci¢city; Isochrysis galbana
Much interest is currently being focused on the very long
chain polyunsaturated fatty acids (PUFAs) arachidonic acid
(AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) because of their involvement in early human retinal
and brain development and disease prevention [1^5]. Their
role as precursors of eicosanoids, a family of biological e¡ec-
tors involved in in£ammatory responses, regulation of blood
pressure and blood clotting, has also been established .
They are obtained either directly from the diet or synthesised
from the g6 and g3 essential fatty acids linoleic acid (LA,
C18:2n-6, v9,12) and K-linolenic acid (ALA, C18:3n-3,
v9,12,15), respectively, by alternating desaturation and elon-
gation reactions. Fatty acid elongation is a four-step process
involving condensation, reduction, dehydration and a second
reduction reaction, with substrate speci¢city residing in the
rate-limiting condensation reaction . This means that elon-
gases with di¡erent substrate speci¢cities can be assembled
using variable condensing components and common dehydra-
tion and reduction components. Studies on the mouse LCE
gene indicated that it encodes the condensation component of
a long-chain fatty acid elongase , and related genes have
been isolated from a number of sources including yeast [9,10],
Mortierella alpina  human , nematodes  and moss
. The characterisation of a C18-v9-speci¢c PUFA elongase
condensation component from Isochrysis galbana , togeth-
er with the isolation of v8 and v4 desaturases from Euglena
 and Thraustochytrium , suggested that EPA and DHA
may be synthesised from ALA by a so-called g3-v8 pathway
in these organisms . In this pathway, which is shown in
Fig. 1, ALA is ¢rst elongated by a C18-v9-speci¢c fatty acid
elongase to C20:3n-3 (g3-eicosatrienoic acid (EtrA)), with
further desaturations/elongations yielding C20:5n-3 (EPA)
and ¢nally C22:6n-3 (DHA).
Alignment of the amino acid sequences of fatty acid elon-
gase condensation components reveals the presence of a
highly conserved histidine-rich motif, HXXHH (‘his-box’)
(Fig. 2). In IgASE1, however, glutamine (Q) replaces the ¢rst
histidine in the his-box, and the signi¢cance of this substitu-
tion is unclear . Similarly, alignment of most membrane-
bound fatty acid desaturases reveals three highly conserved
his-boxes, and these have been implicated in the binding of
di-iron, a requirement for catalytic activity . Site-directed
mutagenesis of these histidine residues resulted in enzyme in-
activation [19,20]. In the so-called ‘front-end’ desaturases
from plants, animals and fungi, the ¢rst histidine in the third
his-box is also substituted with glutamine. Site-directed muta-
genesis of this glutamine in the v6-fatty acid desaturase from
borage resulted in a complete loss of enzyme activity even
when it was replaced by histidine .
We have now assessed the importance of the conserved
histidine box present in the elongase component, particularly
the H to Q substitution found in IgASE1 of I. galbana. Using
a polymerase chain reaction (PCR)-based site-directed muta-
genesis strategy the glutamine in the histidine box of IgASE1
was converted to histidine, alanine or phenylalanine. Further
changes generated the HVYHH sequence found in GLELO1
from Mortierella alpina, which is speci¢c for elongating the
two v6-desaturated PUFAs, Q-linolenic acid (GLA, C18:3n-6)
and stearidonic acid (STA, C18:4n-3) .
2. Materials and methods
2.1. Mutagenesis by PCR
The ampli¢cation of the IgASE1 coding region from an I. galbana
0014-5793/03/$22.00 ? 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
*Corresponding author. Fax: (44)-117-9257374.
E-mail address: email@example.com (B. Qi).
Abbreviations: PUFA, polyunsaturated fatty acid; AA, arachidonic
acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid;
LA, linoleic acid; ALA, K-linolenic acid; GLA, Q-linolenic acid;
STA, stearidonic acid; EDA, g6-eicosadienoic acid; EtrA, g3-eicosa-
FEBS 27427 FEBS Letters 547 (2003) 137^139
cDNA library and its cloning into plasmid pCR2.1-TOPO (Invitro-
gen) is described in Qi et al. . To change the his-box from
QAFHH to HAFHH, ¢rst round PCRs were carried out with Pfu
polymerase (Promega) using primer pairs M13 reverse (universal
primer located on the vector) with GlnHisFor (5P-C TCC TTT CTC
CAT GCC TTC CAC CAC-3P, and M13 forward with GlnHisRev
(5P-GTG GTG GAA GGC ATG GAG AAA GGA G-3P). A second
round PCR, containing 1 Wl of each of the gel-puri¢ed ¢rst-round
products, M13 forward and reverse primers and the Expand High
Fidelity enzyme (Roche), was used to assemble the mutated IgASE1
coding region. The same techniques were used to change glutamine
to alanine (QAFHHCAAFHH) or phenylalanine (QAFHHC
FAFHH), alanine/phenylalanine to valine/tyrosine (QAFHHCQ-
VYHH) and glutamine/alanine/phenylalanine to histidine/valine/tyro-
sine (QAFHHCHVYHH). The primers used are listed in Table 1. All
PCR reactions were carried out under the following conditions: one
initial denaturation step of 94‡C for 3 min; 10 cycles of 94‡C for 15 s,
55‡C for 30 s and 72‡C for 90 s; 20 cycles of 94‡C for 15 s, 55‡C for
30 s, and 72‡C for 93 s (increasing by 3 s in each successive cycle); one
¢nal extension step of 72‡C for 6 min.
Second round PCR products were cloned using the TOPO TA
cloning system (Invitrogen) and the desired base changes con¢rmed
by sequencing. Modi¢ed IgASE1 coding regions were excised from the
pCR2.1-TOPO vector using KpnI and EcoRI and ligated into the
corresponding restriction sites of the yeast expression vector pYES2
(Invitrogen), downstream of the GAL1 promoter. Subsequent yeast
transformation and feeding experiments were performed according
to Qi et al. .
2.2. Fatty acid analysis
Yeast cells were pelleted, washed and dried under a stream of N2.
Total fatty acids were extracted and transmethylated with methanolic
HCl. The fatty acid methyl esters were analysed by gas chromatog-
raphy (GC) on a 30 mU0.25 mm fused silica DB-23 capillary column
(JpW Scienti¢c) using heptadecanoic acid as internal standard and
quanti¢ed by £ame ionisation detection (FID). The chromatograph
was programmed for an initial temperature of 140‡C for 5 min fol-
lowed by a 20‡C/min temperature ramp to 185‡C and a secondary
ramp of 1.5‡C/min to 220‡C. The ¢nal temperature was maintained
for 2 min. Injector and detector temperatures were maintained at
230‡C and 250‡C respectively. The initial head pressure of the carrier
gas (He) was 90 kPa; a split injection was used.
3. Results and discussion
3.1. Functional analysis of wild-type IgASE1
Plasmid pY2ASE1 contains the IgASE1 open reading frame
(ORF) under the control of the GAL1 promoter in the yeast
expression vector pYES2. Transformed yeast cells harbouring
this plasmid were grown on minimal medium supplemented
with LA and ALA to compensate for the lack of endogenous
substrates for the C18-v9-speci¢c elongase. After 48 h the
fatty acid elongation products g6-eicosadienoic acid (EDA,
20:2n-6) and EtrA (20:3n-3) accumulated to 13.6 and 14.8
mol% of total fatty acids, representing some 55 and 48% con-
version of C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3,
respectively (Table 2). These data are consistent with our pre-
vious assays , where we also showed no elongase activity
with GLA (20:3n-6) and clearly demonstrate, therefore, that
the IgASE1 gene encodes a C18-v9-speci¢c PUFA elongating
3.2. Mutagenesis of the IgASE1 his-box
Glutamine in the IgASE1 his-box was replaced by histidine,
alanine or phenylalanine, and the mutant proteins were as-
sayed for activity in transformed yeast. Table 3 shows that
all the substitutions resulted in lower elongase activity,
although complete inactivation was never achieved. The great-
est activity was obtained with the alanine substitution, where
more than 70% of the original enzyme activity was retained.
The phenylalanine substitution had the lowest (but still mea-
surable) activity, whilst the histidine substitution resulted in
an activity that was some 50% of the control value. The glu-
tamine residue in the histidine box thus appears to be essential
for optimum enzyme catalysis, although its substitution did
not result in complete enzyme inactivation. This is in contrast
to the e¡ect of similar mutagenic changes to the glutamine
residue in the third his-box of a v6 desaturase from borage,
which resulted in complete loss of enzyme activity . Step-
wise mutagenesis was also performed to change residues in the
IgASE1 his-box (QAFHH) to match those found in the con-
sensus his-box sequence of several other PUFA-speci¢c elon-
gases, including that of the GLA-speci¢c elongase GLELO1
from Mortierella (HVYHH) (Fig. 2). The double mutant, in
which the middle two amino acids alanine and phenylalanine
in the histidine box were converted to valine and tyrosine but
with the glutamine remaining unchanged (QAFHHCQ-
VYHH), reduced the activity to less than 30% of the wild-
type activity. The triple mutant, in which the ¢rst three amino
acids in the histidine box, QAF, were replaced by HVY
(QAFHHCHVYHH), had less than 10% of the wild-type
Fig. 1. Biosynthesis of EPA and DHA via the g3 (v8) desaturation pathway by some microalgae. In this pathway, ALA is ¢rst elongated by a
C18-v9-speci¢c fatty acid elongase (elo1) to C20:3n-3 (EtrA). A v8 desaturase is required to add a double bond at the v8 position of the car-
bon chain to generate C20:4n-3 (ETA). Further desaturation by a v5 desaturase results in EPA (20:5n-3). DHA (C22:6n-3) is the product of
one more elongation step (elo2) plus a v4 desaturation step.
Fig. 2. Alignment of ‘his-box’ regions of the predicted protein sequence of I. galbana IgASE1  with sequences predicted from other known
PUFA-speci¢c elongating activity genes. GLELO1 is from Mortierella alpina ; HELO1  is from human; PSE1 is from Physcomitrella
patens ; F56H11.4 is from Caenorhabditis elegans .
B. Qi et al./FEBS Letters 547 (2003) 137^139
activity. These results suggest that interactions between the
glutamine and its adjacent amino acids are important for
maximising enzyme activity. The various his-box-mutated
constructs were also expressed in the presence of numerous
other exogenous fatty acid substrates including GLA, STA
and EPA, but no elongation products were observed (data
The results suggest that the his-box is important for catal-
ysis and that all the di¡erences between the his-boxes of other
elongases and IgASE1 are required for optimum enzyme ac-
tivity. However, the elements required to regulate substrate
speci¢city remain to be identi¢ed.
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Oligonucleotide primers used in PCR reactions to construct site-di-
rected mutants of IgASE1/pCR2.1-TOPO
The changed nucleotide bases are indicated by bold type, and the
corresponding amino acids are indicated by bold, italic type and
underlined. The mutated ORFs were con¢rmed by sequencing. The
plasmids were cloned into pYES2 and the resulted constructs were
used for yeast transformation and feeding experiments.
Elongation products of di¡erent fatty acid substrates supplied to yeast cells transformed with the empty vector pYES2 and pY2ASE1
Fatty acidMol% of total fatty acids
3substrate+LA (18:2n-6) +ALA (18:3n-3)
3substrate +LA (18:2n-6)+ALA (18:3n-3)
Exogenous fatty acids supplied as substrates for elongation are indicated by an asterisk. The values given are expressed as mol% of total fatty
acid methyl esters identi¢ed by GC and FID. In the case of elongated substrates, this is also expressed as a per cent elongation (product/pro-
duct+substrateU100). Expression of the IgASE1 transgene was induced by the addition of galactose to yeast cultures. Only C18 substrates
with a double bond at the v9 position were elongated by IgASE1. All values are the means of triplicates from three separate experiments.
Relative conversion of exogenously supplied LA (C18:2n-6) and ALA (C18:3n-3) by wild-type and mutant IgASE1 expressed in yeast
Exogenous fatty acids+18:2+18:3+18:2+18:3
Conversion (%) 55 483120
% IgASE1 10010061 42
B. Qi et al./FEBS Letters 547 (2003) 137^139