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Trypanosoma brucei
oleate desaturase may use a cytochrome
b5
-like
domain in another desaturase as an electron donor
Guillermo A. Petrini, Silvia G. Altabe and Antonio D. Uttaro
Instituto de Biologı
´
a Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiologı
´
a, Facultad de Ciencias
Bioquı
´
micas y Farmace
´
uticas, Universidad Nacional de Rosario, Santa Fe, Argentina
An open reading frame with fatty acid desaturase similarity
was identified in the genome of Trypanosoma brucei.The
1224 bp sequence specifies a protein of 408 amino acids with
59% and 58% similarity to Mortierella alpina and Arabid-
opsis thaliana D12 desaturase, respectively, and 51% with
A. thaliana x3 desaturases. The histidine tracks that com-
pose the iron-binding active centers of the enzyme were
more similar to those of the x3 desaturases. Expression of
the trypanosome gene in Saccharomyces cerevisiae resulted
in the production of fatty acids that are normally not syn-
thesized in yeast, namely linoleic acid (18:2D9,12) and
hexadecadienoic acid (16:2D9,12), the levels of which were
dependent on the culture temperature. At low temperature,
the production of bi-unsaturated fatty acids and the 16:2/
18:2 ratio were higher. Transformed yeast cultures supple-

mented with 19:1D10 fatty acid yielded 19:2D10,13, indica-
ting that the enzyme is able to introduce a double bond at
three carbon atoms from a pre-existent olefinic bond. The
expression of the gene in a S. cerevisiae mutant defective in
cytochrome b5 showed a significant reduction in bi-unsat-
urated fatty acid production, although it was not totally
abolished. Based on the regioselectivity and substrate pre-
ferences, we characterized the trypanosome enzyme as a
cytochrome b5-dependent oleate desaturase. Expression of
the ORF in a double mutant (ole1D,cytb5D)abolishedall
oleate desaturase activity completely. OLE1 codes for the
endogenous stearoyl-CoA desaturase. Thus, Ole1p has, like
Cytb5p, an additional cytochrome b5 function (actually
an electron donor function), which is responsible for the
activity detected when using the cytb5D single mutant.
Keywords: fatty acids; desaturation; electron donor; cyto-
chrome; trypanosomatids.
Trypanosomatids are parasitic protozoa that belong to
the order Kinetoplastida. They are the causative agents of
several highly disabling and often fatal diseases occurring in
tropical and subtropical parts of the world, which include
human African sleeping sickness and the related cattle
disease Nagana, both caused by Trypanosoma brucei
subspecies. It is estimated that there are 300 000–500 000
cases of human sleeping sickness per year, which are fatal
if untreated [1].
The drugs used in the treatment of trypanosomiasis are
toxic and in some cases have low effectiveness. This makes
the development of new chemotherapeutic compounds
against these diseases urgent [1].

Trypanosomatids contain the usual range of lipids found
in eukaryotes. Although the fatty acid composition of
bloodstream trypanosomes is, in several respects, similar to
that of lipids found in the plasma of their mammalian host,
some essential differences suggest that trypanosomes can
regulate their fatty acid composition. T. brucei possesses
a higher proportion of linoleic acid (18:2D9,12) and other
polyunsaturated fatty acids (PUFAs) such as 22:5 and 22:6,
and lower levels of oleate (18:1D9) and C16 fatty acids, as
compared with the plasma lipid fatty acids of the human
host [2,3].
The presence of these molecules suggests that fatty acid
desaturation occurs via the so-called Ôplant pathwayÕ where
double bonds are introduced toward the methyl end of
the molecule. In mammals, by contrast, double bonds are
always introduced toward the carboxyl end of the fatty acid
molecule [4].
Membrane fluidity is of central importance for the
function and integrity of the membrane system of the cell.
It is essential for the mobility and function of embedded
proteins and for forming membrane curvatures, which
in turn are required for the formation of organelles, the
vesicular system and the nuclear envelope. A crucial
parameter that determines membrane fluidity is the
balance between saturated and unsaturated fatty acids
(UFAs) [4,5].
Poikilothermic organisms possess the potential ability to
modify the fatty acyl composition of their membrane
phospholipids in response to changes in environmental
Correspondence to A. D. Uttaro, IBR-CONICET, Depto.

Microbiologı
´
a, Facultad de Ciencias Bioquı
´
micas y Farmace
´
uticas,
Universidad Nacional de Rosario, Suipacha 531,
2000-Rosario, Santa Fe, Argentina.
Fax: + 54 341 4390465, Tel.: + 54 341 4350661,
E-mail:
Abbreviations: UFA, unsaturated fatty acids; PUFA, polyunsaturated
fatty acids; FAME, fatty acid methyl ester; GSS, genome survey
sequences.
Database: The nucleotide sequence reported in this paper has been
submitted to the GenBank
TM
/EBI Data Bank with accession number
AY372529.
(Received 26 September 2003, revised 19 December 2003,
accepted 20 January 2004)
Eur. J. Biochem. 271, 1079–1086 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04005.x
temperature [6,7]. The general trend is an increase in UFAs
at lower growth temperatures and an increase in saturated
fatty acids at higher temperatures. Such compositional
adaptation of membrane lipids, which is called a homeo-
viscous adaptation process [8], serves to maintain the correct
membrane fluidity at the new conditions.
Temperature changes are also experienced by a trypano-
somatid parasite when it leaves the insect and enters the

tissues of a vertebrate host. It has been shown that
differentiation of in vitro cultured mammalian-stage to
insect-stage Trypanosoma and Leishmania sp. can be trig-
gered by a temperature shift [9]. Differentiation of these
parasites involves dramatic changes in the shape of the cells
and the morphology of some organelles. It seems probable
that membrane fluidity plays an important role in estab-
lishing these morphological alterations.
The fact that high membrane fluidity is possibly essential
for trypanosome transmission, together with the observed
differences in the degree and type of fatty acid desaturation
between trypanosomes and their mammalian host, indicate
that fatty acid desaturases may be good targets for
trypanocidal drugs.
Fatty acid desaturases are nonheme iron-containing
oxygen-dependent enzymes involved in the regioselective
introduction of double bonds in fatty acyl aliphatic chains.
Three classes of regioselectivity have been observed. The Dx
desaturases introduce a double-bond x-carbons from the
carboxyl end; xx desaturases introduce a double-bond
x-carbons from the methyl end; and the Ôm +xÕ desaturases
introduce a double-bond x-carbons from an existing double
bond [10].
The desaturation pathway starts by the introduction of
a double bond between C9 and C10 of stearoyl-ACP (in
plants) or stearoyl-CoA (in yeast and animals), producing
oleoyl-thioesters. Further desaturation occurs on fatty acyl
chains of phospholipids, as in plants, where an oleate or D12
desaturase produce linoleic acid (18:2D9,12). Mammals are
unable to synthesize linoleic acid but incorporate this

essential PUFA from dietary sources [4].
D12 fatty acid desaturase genes have been isolated from
several species of cyanobacteria, fungi and plants including
Arabidopsis, soybean and parsley [11]. The encoded enzymes
are all believed to be integral membrane proteins utilizing
an acyl-lipid substrate, and with the exception of the
cyanobacterial and plastidial enzymes, requiring cyto-
chrome b5 for the electron transport. The deduced amino
acid sequences of these desaturases show a good deal of
similarity, most notably in the region of the three histidine-
rich motifs present in all desaturases, which are presumed to
comprise the iron-binding active centers of the enzyme
[12,13].
In this work we describe the isolation and functional
characterization of a T. brucei oleate desaturase by hetero-
logous expression in S. cerevisiae. This is, to our knowledge,
the first report on the isolation of a desaturase from
trypanosomatids, and one of the few reported for such an
enzyme from protozoa. As this activity is not present in
mammals it could be a relevant target for the design of
drugs useful in chemotherapy. A detailed study of the
biochemical properties of the parasite’s oleate desaturase
allowed us to identify a novel alternative electron donor for
the desaturase reaction.
Experimental procedures
Materials
Cis-10-nonadecenoic (9:1D10), gondonic (cis 20:1D11),
erucic (cis 22:1D13), oleic (cis 18:1D9), linoleic (18:2D9,12),
petroselinic (cis 18:1D6), and vaccenic (cis 18:1D11) acids
(all > 99% pure), Tergitol NP-40, dimethyl disulfide,

sodium methoxide, yeast nitrogen base, glucose and amino
acids were obtained from Sigma. All solvents were
purchased from Merck.
Cloning, sequencing and sequence analysis
Procyclic trypanosomes (strain 427) were grown in SDM-79
medium [14] and genomic DNA was prepared by standard
methods. Two regions of the T. brucei genome were
amplified using forward and reverse primers designed on
the ends of single pass sequences TF and TR, from genome
survey sequence (GSS) 35I5 (respectively: 5¢-CATGTCAC
GGCTAAGGTAGC-3¢ and 5¢-CTAAGCAACAGATGG
GAGGT-3¢)andGSS38K3(5¢-CCAACGCACCGTTCT
TTCG-3¢ and 5¢-ACTGCGAGTAATGCAGATCC-3¢)
identifiedintheT. brucei genome database of TIGR
(). These fragments were cloned in
Escherichia coli using the pGEM-T Easy vector (Promega)
by using standard methods and were sequenced completely.
It allowed us to cover a region of the genome containing an
ORF with desaturase similarity. A 1227 bp genomic clone
was obtained by PCR amplification with the forward primer
5¢-CG
GGATCCATGTTGCCTAAGCAACAGATG-3¢
and the reverse primer 5¢-CCC
AAGCTTAACTGCGAG
TAATGCAGAT-3¢ containing BamHI and HindIII sites,
respectively (underlined), designed on the ORF regions
coding for the predicted N-terminal and C-terminal ends of
the polypeptide. Amplifications involved an initial denatur-
ationstepat94°C for 4 min followed by 30 cycles of
denaturation at 94 °C for 1 min, annealing at 58 °Cfor

1 min, and extension at 72 °C for 2 min. The 1.2 kb
product was ligated into the pGEM-T Easy vector, cloned
and the nucleotide sequence determined. Amino acid
sequences were aligned by using
CLUSTALX
[15]. Hydropathy
profile analysis and prediction of transmembrane regions
were performed using the
TMPRED
program available
online at />form.html [16].
Expression of the
T. brucei
desaturase gene
The cloned sequence was ligated into the BamHI and
HindIII sites of p426GPD, the 2-micron yeast expression
vector containing a glyceraldehyde-3-phosphate dehydro-
genase promoter [17]. This vector contains a selectable
marker gene, which confers uracil prototrophy in the host.
The resulting plasmid construct, pDes12, and the vector
alone were transformed by electroporation into S. cerevisiae
strain HH3 [18] and mutant yeast strains kindly provided
by C. E. Martin [19] (Table 1 shows relevant genotypes).
Transformed yeasts were selected on minimal agar plates
lacking uracil [20].
To determine the enzyme activity at different tempera-
tures, transformed yeast strains were cultured overnight at
1080 G. A. Petrini et al.(Eur. J. Biochem. 271) Ó FEBS 2004
30 °C in 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose
and leucine, tryptophan, adenine, lysine and histidine (all at

20 mgÆL
)1
) if required. The cultures then were diluted to a
D
600
value of 0.2 and grown for 72 h in a shaking incubator
at 30 °C, 25 °Cand20°C. Fatty acid supplements were
added to the cultures as solutions in ethanol to a final
concentration of 1 m
M
, plus 0.1% (v/v) of Tergitol NP-40.
Translational arrest was performed by adding cyclohex-
imide at a final concentration of 0.5 mgÆmL
)1
toa30mL
culture grown at 30 °C. The culture was immediately
divided into three subcultures of 10 mL each and incubated
at different temperatures for 72 h. Controls of growth and
protein synthesis arrest were carried out by assaying the
D
600
and following the radioactive labeling of proteins with
[
32
S]methionine.
Fatty acid analysis
Cells from 20 mL cultures were collected by centrifugation
at 500 g for 5 min, and the pellets washed twice with 20 mL
of distilled water. Lipids were extracted according to Bligh
and Dyer [21]. The organic phase was reduced to dryness

under N
2
, and fatty acid methyl esters (FAMEs) were
prepared by adding 1 mL of 0.5
M
sodium methoxide in
methanol and incubating for 20 min at room temperature.
After neutralization with 6
M
HCl and extraction with 2 mL
hexane, the organic solvent was evaporated to dryness
under an N
2
stream.
FAME composition was analysed with a polyethylene
glycol column (WAX, 30 m · 0.25 mm inside diameter,
Perkin Elmer) in a Perkin Elmer AutoSystem XL gas
chromatograph. Gas chromatographic analysis was per-
formed at 180 °C isothermically. The GC-MS was carried
out using a Perkin Elmer mass detector (model TurboMass)
operated at an ionization voltage of 70 eV with a scan range
of 20–500 Da. The retention time and mass spectrum of any
new peak obtained was compared with that of standards
(Sigma) and those available in the data base NBS75K
(National Bureau of Standards). For double bond posi-
tional analysis, the FAMEs were derivatized with dimethyl
disulfide and the adducts analysed by GC-MS as described
previously [22].
Results
Cloning and structural characterization of an oleate

desaturase from
T. brucei
A BLAST search carried out using databases from trypan-
osomatid genome projects identified two T. brucei GSS with
high similarity to fatty acid desaturases. The deduced amino
acid sequences from GSS 35I5.TF and GSS 38K3.TF
showed 59–63% similarity to a central portion of x6
desaturase from Arabidopsis thaliana.ByPCRusing
oligonucleotides designed at the end of the forward (TF)
and reverse (TR) sequences of GSSs we amplified and
subsequently sequenced a region of the T. brucei genome
covering 2.5 kb. It contains an ORF of 1227 bp that codes
for a putative protein of 408 amino acids, with a number of
characteristic features of fatty acid desaturases including
three histidine boxes supposed to constitute the iron-binding
active centers of the enzyme [12,13]. Interestingly, the third
histidine box presents characteristics that could be ascribed
bothtoaD12 or x3 desaturase [12,13], as it has two
additional histidines at a distance of four amino acids
upstream of the consensus motif H-X
2
-H
2
(Fig. 1). No
consensus sequences for a cytochrome b5-like domain were
detected [19].
Alignment of the amino-acid sequences of known acyl
lipid desaturases revealed that the T. brucei desaturase
candidate possesses 43% identity and 59% similarity to
Mortierella alpina D12 desaturase (accession Q9Y8H5),

40% identity and 58% similarity to A. thaliana x6desatu-
rase (P46313), 32% identity and 51% similarity to endo-
plasmic reticulum x3desaturasefromA. thaliana (P48623),
and 30% identity and 51% similarity to chloroplastic x3
desaturase from A. thaliana (P46310).
In a new BLAST search carried out after the update of
the genome databank at TIGR, using the ORF sequence
as a query, a sequence annotated as a putative T. brucei
x6 desaturase was detected with accession number
AC007862 : 100803–102106. This sequence is 99% identical
to our T. brucei desaturase candidate and was located on
chromosome II of T. brucei stock TREU927 GUTat10.1.
Only two differences with the sequence determined by us
were noted in the sequence from the database, namely
Val124 and Thr406 (Fig. 1) instead of isoleucine and
tyrosine, respectively. These differences should probably
be attributed to strain-dependent sequence variations, as we
used T. brucei 427 stock in this study.
The hydropathy profile of the T. brucei desaturase was
compared to that of the endoplasmic reticulum x6desatu-
rase of A. thaliana (data not shown). The predicted
transmembrane topology appears to be similar to that of
other desaturases, with two long hydrophobic domains,
each spanning the membrane twice [12].
Functional characterization of the
T. brucei
desaturase
gene in
S. cerevisiae
Functional characterization was carried out by determining

the fatty acid profiles of S. cerevisiae transformed either
with vector p426GPD alone or the vector with a DNA
insert harboring the putative T. brucei desaturase (pDes12).
Table 1. Relevant genotype of yeast strains used and source references.
Strain Genotype Source
HH3 MATa, trp1–1, ura3–52, ade2–101, his3–200, lys2–801, leu2–1 [18]
AMY-1a MATa, cytb5::LEU2, OLE1, TRP1, can1–100, ura3–1, ade2–1, his3–11, his3–15 [19]
AMY-3a MATa, CYTB5, ole1(DHpa)::LEU2, trp1–1, can1–100, ura3–1, ade2–1, HIS3 [19]
AMY-5a MATa, cytb5::LEU2, ole1(DBstEII)::LEU2, ura3–1, ade2–1 [19]
Ó FEBS 2004 Trypanosoma brucei oleate desaturase (Eur. J. Biochem. 271) 1081
The fatty acid composition of the yeast transformed
with p426GPD showed the four main fatty acids normally
found in S. cerevisiae, namely 16:0, 16:1D9, 18:0 and 18:1D9
(Fig. 2A). This result is consistent with the fact that
S. cerevisiae does not possess D12 desaturase activity [23].
Additional peaks were observed in the profiles of pDes12-
transformed yeast (Fig. 2B,C). Based on GC retention times
and mass spectra, the additional peaks associated with the
presence of the T. brucei gene, indicated in Fig. 2, were
identified as 16:2D9,12 and 18:2D9,12, respectively. This
indicates that the T. brucei desaturase gene was functionally
expressed in the yeast cells and acted on the endogenous
monounsaturated substrates to give 16:2 and 18:2 PUFAs.
To confirm the position of the double bonds created by
the T. brucei desaturase gene product in yeast, the FAME
samples were converted to dimethyl disulfide adducts and
analysed by GC-MS. The mass spectrum of the 18:2
adducts showed a weak ion at m/z 388 corresponding to the
theoretical mass for the molecular ion of the dimethyl
disulfide adducts (Fig. 3). When two methylthio groups

were introduced to the double bond at the C-9,10 or C-12,13
carbons of 18:2D9,12 methyl esters, a set of key fragment
ions at m/z 171, 185, 217, 293 [M-(SCH
3
)
2
-H] and 340
[M-(SCH
3
)-H] for the isomer I [methyl 9,10-bis(methyl-
thio)octadec-12-enoate (Fig. 3A)] and m/z 131, 225, 257,
293 and 340 for the isomer II [methyl 12,13-bis(methylthio)-
octadec-9-enoate (Fig. 3B)] was obtained [22]. These results
indicate the presence of double bonds at the D9 and D12
positions. Similar data (not shown) identify the 16:2 as a
D9,12 fatty acid.
The accumulation of bi-unsaturated fatty acids in the
HH3/pDes12 transformants was investigated at different
temperatures (Fig. 2B,C and Table 2). The relative amount
of bi-unsaturated fatty acids was found to increase in HH3/
pDes12 with decreasing temperatures. Furthermore, the
ratio 16:2/18:2 was 0.055 at 30 °C, 0.14 at 25 °C and 0.21 at
20 °C. These results indicate that the T. brucei desaturase
activity and its substrate specificity change with the growth
temperature. To rule out the possibility that it could be an
effect of increased synthesis of the plasmid born desaturase
we repeated the experiment in the presence of a translation
inhibitor. A culture of HH3/pDes12 was grown at 30 °Cto
near stationary phase (D
600

¼ 1.5). Cycloheximide was then
added and the culture immediately divided into three flasks,
with identical culture volume. Each flask was incubated at
30 °C, 25 °Cand20°C, respectively, for three days, and
FAMEs were analysed as before. As shown in Table 2, a
similar increase in bi-unsaturated fatty acids and 16:2/18:2
ratio was found at lower temperatures.
Substrate preference and regioselectivity
AsindicatedinTable2,T. brucei desaturase shows maxi-
mum activity with oleate as deduced from the rates of
conversion for mono- to bi-unsaturated fatty acids. In order
to further characterize the substrate preference and regio-
chemistry of T. brucei desaturase, cultures of transformed
yeast were supplemented with different fatty acids and the
FAME profile of the extracted lipids analysed as before.
Erucic acid (22:1D13) and gondonic acid (20:1D11) were
poorly incorporated into yeast membranes (less than 0.5%
of total FAMEs) and no bi-unsaturated fatty acids derived
from them were detectable. Vaccenic acid (18:1D11),
petroselinic acid (18:1D6), and 19:1D10 fatty acid were
incorporated at moderate levels (15%, 18% and 12%,
respectively) into the yeast lipids. Only 19:1 was converted
(8.6% of conversion at 20 °C) to the corresponding 19:2
fatty acid in HH3/pDes12. The GC-MS analysis of the
Fig. 1. Alignment of deduced amino acid sequence of T. brucei oleate desaturase with other membrane-bound desaturase sequences. Identical residues
are indicated by white type on grey shading. The three histidine-rich domains are indicated I–III. A. thaliana, D12-desaturase from Arabidopsis
thaliana (P46313); M. alpina, Mortierella alpina D12-desaturase (Q9Y8H5). The changes in amino acids V124I and T406Y between T. brucei strains
426 and TREU927 are indicated, with I and Y below the sequence, respsectively.
1082 G. A. Petrini et al.(Eur. J. Biochem. 271) Ó FEBS 2004
FAME dimethyl disulfide adduct showed two additional

peaks corresponding to the isomer I [methyl 10,11-bis
(methylthio) nonadec-13-enoate] and isomer II [methyl
13,14-bis (methylthio) nonadec-10-enoate] of 19:2 adducts.
These compounds showed the ion at m/z 402 corresponding
to the molecular ion and fragmentation products 171, 199,
231, 307 [M–(SCH
3
)
2
–H] and 358 (M–SCH
3
–H) for isomer
I (Fig. 4A) and 131, 239, 271, 307 and 358 for isomer II
(Fig. 4B). This confirms that 19:2 is a D10,13 bi-unsaturated
fatty acid, indicating that the T. brucei desaturase possesses
the ability to introduce a double bond at three carbon atoms
counting from a pre-existent olefinic bond.
Characterization of electron donors
To gain information about the possible electron donor
for the desaturase reaction, we transformed a cytochrome
b5-deficient yeast mutant (S. cerevisiae AMY-1a strain,
Table 1) with p426GPD or pDes12 and their FAMEs were
analysed by GC-MS.
The two additional peaks corresponding to 16:2 and
18:2 fatty acids, as observed in spectra taken with FAMEs
prepared from wild-type yeast cells transformed with the
T. brucei gene (HH3/pDes12), and which are absent from
those prepared with cells not having trypanosome enzyme
(AMY-1a/p426GPD) (see above and Table 2), can still be
seen in cytochrome b5 mutant cells in which the trypano-

some desaturase was expressed (AMY-1a/pDes12). How-
ever, in this mutant, the amounts of the bi-unsaturated
compounds were threefold lower than in the transformed
wild-type yeast cells (Table 2). This result indicates that the
endogenous diffusible cytochrome b5 is active in transfer-
ring electrons to the oleate desaturation reaction but that an
Fig. 2. Identification of fatty acid desaturation products in transformed
yeasts. GC analysis of FAMEs from yeast (HH3) transformed with
p426GPD grown at 30 °C(A)andwithpDes12grownat30°C(B)or
20 °C (C). Peaks corresponding to relevant fatty acids are indicated:
16:0, palmitate; 16:1, palmitoleate; 16:2, D9,12-hexadecadienoate; 18:0,
stearate; 18:1, oleate; 18:2, linoleate.
Fig. 3. Mass spectra of 18:2 adducts. Dimethyl disulfide adducts were
prepared from FAME extracted from yeast transformed with pDes12
grown at 20 °C and analysed by GC-MS as described previously [22].
(A) Isomer I of 18:2 adduct (methyl 9,10-bis(methylthio)octadec-12-
enoate); (B) isomer II (methyl 12,13-bis(methylthio) octadec-9-enoate).
Key fragments are indicated.
Ó FEBS 2004 Trypanosoma brucei oleate desaturase (Eur. J. Biochem. 271) 1083
alternative electron donor, also present in yeast, can do so as
well. We speculate that it could be the cytochrome b5
domain of Ole1p, the bifunctional yeast protein representing
the stearoyl-CoA desaturase [19]. To test this theory we
transformed the doubly disrupted strain AMY-5a (ole1D::
LEU2, cytb5D::LEU2) with pDes12 and grew it in the
presence of oleate. The analysis of the FAME profile
showed that oleate was incorporated into the cell lipids in a
proportion representing 60% of the total fatty acids
(Table 2). No other UFA was detected.
To be sure that the exogenous oleate was correctly

incorporated into the cell phospholipids and accessible
totheoleatedesaturase,thetransformedwildtypestrain
(HH3/pDes12) was grown in the presence of oleate. The
oleate found in the cell lipids amounts to 41% of the
FAMEs (Table 2). This percentage represents both the fatty
acid derived from the incorporated, exogenous oleate and
the material de novo synthesized by the cell (endogenous
source). Thirty percent of the oleate (18:1) appeared to have
been converted to linoleate (18:2), similar to that observed
previously for cells not grown in the presence of exogenous
oleate.
In an additional control experiment, the singly disrupted
strain AMY-3a (ole1D::LEU2)wastransformedwith
pDes12. As shown in Table 2, AMY-3a/pDes12 incorpor-
ated exogenously added oleate at a level comparable to that
of the transformed double mutant, but converted it to
linoleate (18:2) up to 27%. This conversion rate is similar to
that of the wild type transformed strain using the endo-
genous or exogenous oleate as substrate. This indicates that
exogenous substrate is accessible to oleate desaturase in the
double mutant, but was not desaturated due to the absence
of appropriate electron donors for the reaction.
Discussion
De novo synthesis of fatty acids was recently proved to be
present in African trypanosomes [24,25], although for many
years it was believed to be absent or at very low activity in all
trypanosomatids [3]. These parasites can efficiently take up
free fatty acids from the medium. Even though this uptake
Table 2. Incorporation of exogenous fatty acids and conversion of mono- to bi-unsaturated fatty acids in transformed yeasts. Incorporation expressed
as a percentage of total fatty acids. Cyc, cycloheximide; ND, not detectable; n ¼ 3.

Strain Growth temperature Supplement Incorporation (%)
Conversion (%)
16:2/18:2
16:1 to 16:2 18:1 to 18:2
HH3/pDes12 30 °C – – 1.4 ± 0.2 25 ± 2 0.055 ± 0.005
25 °C – – 3.8 ± 0.4 28 ± 3 0.14 ± 0.02
20 °C – – 6.5 ± 0.6 32 ± 3 0.21 ± 0.03
30 °C Cyc – 0.8 ± 0.1 12 ± 1 0.068 ± 0.008
25 °C Cyc – 1.3 ± 0.1 13 ± 1 0.10 ± 0.02
20 °C Cyc – 1.9 ± 0.2 15 ± 1 0.13 ± 0.02
20 °C 18:1 D9 41 ± 4
a
6.2 ± 0.5 30 ± 3 0.21 ± 0.02
AMY-1a/pDes12 20 °C – – 2.0 ± 0.1 11 ± 1 0.19 ± 0.03
AMY-3a/pDes12 20 °C 18:1 D9 61 ± 4 ND 27 ± 3 –
AMY-5a/pDes12 20 °C 18:1 D9 60 ± 5 ND ND –
a
Endogenous and exogenous oleate.
Fig. 4. Mass spectra of 19:2 adducts. Dimethyl disulfide adducts were
prepared from FAME extracted from yeast transformed with pDes12
grown at 20 °C, supplemented with 19:1 and analysed by GC-MS as
described previously [22]. (A) Isomer I of 19:2 adduct [methyl 10,11-bis
(methylthio) nonadec-13-enoate]; (B) isomer II [methyl 13,14-bis
(methylthio) nonadec-10-enoate]. Key fragments are indicated.
1084 G. A. Petrini et al.(Eur. J. Biochem. 271) Ó FEBS 2004
can account for a big proportion of the fatty acids that
constitute the parasite lipids, it is difficult to explain the high
amount of linoleate and linolenate present in trypanosom-
atids. As these essential fatty acids are present at low levels
in the mammalian hosts, some kind of regulation in fatty

acid composition has to be present in the trypanosome [2].
Evidence of desaturase activities in trypanosomes has
been documented previously. By using radioactive fatty
acids such as stearic or oleic acid, different species of
trypanosomatids have been shown to produce oleate,
linoleate and linolenate [3,26]. Our work represents the first
report about the isolation and functional characterization of
an oleate desaturase from a trypanosomatid. It confirms
that these organisms are able to synthesize linoleic acid, and
as this activity is not present in mammals, oleate desaturase
constitutes a good candidate as a target for chemotherapy.
It is for this purpose that we have characterized its structural
and enzymatic properties.
T. brucei oleate desaturase presents a high degree of
similarity to D12 and x3 desaturases from plants and fungi
(54–51%) with the higher identity to D12 desaturases.
Interestingly, the trypanosomatid enzyme has a conserved
motif at the third histidine box, which is more similar to that
of the x3 desaturases [13]. The functional characterization
allowed us to show that this enzyme is not an x3 desaturase.
This indicates that the H
2
-X
4
-H-X
2
-H
2
motif cannot be
the consensus motif for x3 desaturases [13]. However, the

desaturase described here is strictly a Ôm +xÕ type and not a
D12 or x6 desaturase, as indicated by its ability to convert
19:1D10 into 19:2D10,13. Whether this amino-acid motif
is related to this kind of regioselectivity remains to be
determined.
S. cerevisiae expressing T. brucei oleate desaturase pro-
duces more bi-unsaturated fatty acids at low temperature,
which is the expected behaviour for an enzyme that is
involved in cold adaptation. As our DNA construct is under
a constitutive yeast promoter, it indicates that the tempera-
ture effect is either due to a post-transcriptional regulation,
or that regulation occurs at the enzyme activity level. As low
temperature can have a stimulatory effect on transcription
by increasing negative DNA supercoiling, especially on
plasmid-borne genes, we repeated the experiments in the
presence of the translation inhibitor cycloheximide. Our
results show that the stimulatory effect of low temperature
persists, indicating a direct effect of temperature, or an
indirect one via membrane fluidity, on oleate desaturase
activity itself. Moreover, low temperature appears to
increase the substrate specificity of the enzyme for shorter
chain UFAs (Table 2). Although an increase in the
exogenous desaturase activity due to a decreased proteolytic
degradation at lower temperature could not be ruled out,
it cannot account for the change of substrate specificity.
Unfortunately, the expression level of the oleate desaturase
is very low, as judged from our lack of success with the
detection of recombinant protein by immunological meth-
ods (data not shown).
Two types of electron donors for desaturases have been

described. In cyanobacteria and plastids, the couple ferre-
doxin and ferredoxin-NADP
+
oxidoreductase are involved
in the desaturase reaction. In the endoplasmic reticulum of
plants, animals and fungi the electron flow is controlled by
the small and diffusible cytochrome b5 and cytochrome b5
reductase [13]. S. cerevisiae contains only one fatty acid
desaturating enzyme, the stearoyl-CoA D9 desaturase which
is encoded by OLE1. Interestingly, Ole1p has a cyto-
chrome b5 domain at its carboxy-terminal end [19].
T. brucei oleate desaturase lacks a consensus sequence
for a covalently linked cytochrome b domain, so either a
diffusible cytochrome b5 or ferredoxin should serve as its
electron donor. When expressed in a cytb5D yeast mutant,
the T. brucei oleate desaturase showed only one third of its
activity compared to the protein expressed in wild type
yeast. This indicates that in yeast cytochrome b5 serves as
the major electron donor, but an alternative donor has to
account for the remainder of the activity. The lack of oleate
desaturase activity when expressed in the ole1D, cytb5D
double mutant indicates that the cytochrome b5 domain
of Ole1p is involved in  30% of the electron flow to
desaturase. To our knowledge this is the first time that this
kind of alternative electron transfer has been described.
One strong criticism that might be raised against this last
interpretation is that the exogenous substrate may not be
accessible to the enzyme. As ole1 mutants are auxotrophic
for monounsaturated fatty acids, we complemented them
with oleate. The exogenous oleate could be taken up by the

cells and stored in lipids as triacylglycerols, or esterified
into phospholipids that could be poor substrates for oleate
desaturase. To rule out these possibilities we expressed the
oleate desaturase in an ole1D single mutant. The exogenous
oleate was incorporated into the yeast lipids accounting for
61% of total fatty acids, and 27% was converted into
linoleate. Therefore, this oleate pool and the endogenous
pool in wild type cells are equally accessible to desaturation.
The colocalization of oleate desaturase, cytochrome b5
and Ole1p could indicate that the expressed enzyme is
associated with the endoplasmic reticulum of S. cerevisiae
and this suggests that the same compartmentation would
occur in trypanosomatids. It is interesting to note that we
have detected sequences with a high degree of similarity to
cytochrome b5 and stearoyl-CoA desaturases containing a
C-terminal cytochrome b5 domain in Leishmania major
(data not shown). This suggests that the trypanosomatid
oleate desaturase in its natural environment could interact
with a similar kind of electron donor as is the case in
yeast, probably forming an enzymatic complex with other
desaturases.
In order to validate T. brucei oleate desaturase as a target
for chemotherapy, knock out experiments are in progress in
our laboratory. As it has been observed that a temperature
shift is involved in triggering cellular differentiation in
trypanosomatids [9], a T. brucei strain defective in desatu-
rase activity could be instrumental in determining whether a
variation in membrane fluidity or in fatty acid composition
of the parasite membrane would by itself play an essential
role in triggering the process.

Acknowledgements
We wish to thank Mo
´
nica Hourcade for technical assistance and Fred
R. Opperdoes, Paul A. M. Michels and Diego de Mendoza for
comments and suggestions on the manuscript. We gratefully acknow-
ledge Dr Charles E. Martin and Olga A. Castro for generously
providing us with the yeast strains and to the T. brucei genome project
and The Institute of Genomic Research (TIGR) for the availability of
Ó FEBS 2004 Trypanosoma brucei oleate desaturase (Eur. J. Biochem. 271) 1085
genome survey sequences data. A. D. U. is a member of Carrera del
Investigador Cientı
´
fico, CONICET, Argentina. G. A. P. has a
fellowship from Secretarı
´
a de Ciencia y Tecnologı
´
adelaNacio
´
n
(SECyT), Argentina. This work was supported by Fondo Nacional de
Ciencia y Tecnologı
´
a, SECyT, Argentina, grant PICT 99 N°1–7160.
References
1. World Health Organization (2001) African trypanosomiasis. Fact
Sheet Number 259. WHO Publications, Geneva.
2. Mellors, A. & Samad, A. (1989) The acquisition of lipids by
African trypanosomes. Parasitol. Today 5, 239–244.

3. Haughan, P.A. & Goad, L.J. (1991) Lipid biochemistry of try-
panosomatids. In Biochemical Protozoology (Coombs, G.H. &
North, M.J., eds), pp. 312–328. Taylor & Francis, London.
4.Gurr,M.I.&Harwood,J.L.(1991)Lipid Biochemistry. An
Introduction. Chapman & Hall, London.
5. Braun,S.,Matuschewski,K.,Rape,M.,Thoms,S.&Jentsch,S.
(2002) Role of the ubiquitin-selective CDC48
UFD1/NPL4
chaperone
(segregase) in ERAD of OLE1 and other substrates. EMBO J. 21,
615–621.
6. Macartney, A., Maresca, B. & Cossins, A.R. (1994) Acyl-CoA
desaturases and the adaptive regulation of membrane lipid com-
position. In Temperature Adaptation of Biological Membranes
(Cossins, A.R., ed.), pp. 129–139. Portland Press, London.
7. Sakamoto, T. & Murata, N. (2002) Regulation of the desaturation
of fatty acids and its role in tolerance to cold and salt stress. Curr.
Opin. Microbiol. 5, 206–210.
8. Sinensky, M. (1974) Homeoviscous adaptation – a homeostatic
process that regulates the viscosity of membrane lipids in
Escherichia coli. Proc. Natl Acad. Sci. USA 71, 522–525.
9. Williams, G.T. (1985) Control of differentiation in Trypanosoma
cruzi. Curr. Top. Microbiol. Immunol. 117, 1–22.
10. Shanklin, J. & Cahoon, E.B. (1998) Desaturation and related
modifications of fatty acids. Annu. Rev. Physiol. Plant Mol. Biol.
49, 611–641.
11. Huang, Y S., Chaudhary, S., Thurmond, J.M., Bobik, E.G. Jr,
Yuan, L., Chan, G.M., Kirchner, S.J., Mukerji, P. & Knutzon,
D.S. (1999) Cloning of delta12- and delta6-desaturases from
Mortierella alpina and recombinant production of gamma-lino-

lenic acid in Saccharomyces cerevisiae. Lipids 34, 649–659.
12. Shanklin, J., Whittle, E. & Fox, B.G. (1994) Eight histidine
residues are catalytically essential in a membrane-associated iron
enzime, stearoyl-CoA desaturase, and are conserved in alkane
hydroxilase and xilene monooxygenase. Biochemistry 33, 12787–
12794.
13. Los, D.A. & Murata, N. (1998) Structure and expression of fatty
acid desaturases. Biochim. Biophys. Acta 1394, 3–15.
14. Brun, R. & Schoeneberger, M. (1979) Cultivation and in vitro
cloning of procyclic culture forms of Trypanosoma brucei in a
semi-defined medium. Acta Tropica 36, 289–292.
15. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL
W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–
4680.
16. Hofmann, K. & Stoffel, W. (1993) TMbase – A database of
membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler
374, 166.
17. Mumberg, D., Mu
¨
ller, R. & Funk, M. (1995) Yeast vectors for the
controlled expression of heterologous proteins in different genetic
backgrounds. Gene 156, 119–122.
18. Castro, O., Chen, L.Y., Parodi, A.J. & Abeijo
´
n, C. (1999) Uridine
diphosphate-glucose transport into the endoplasmic reticulum of
Saccharomyces cerevisiae: in vivo and in vitro evidence. Mol. Biol.
Cell 10, 1019–1030.

19. Mitchell, A.G. & Martin, C.E. (1995) A novel cytochrome b5-like
domain is linked to the carboxyl terminus of the Saccharomyces
cerevisiae delta-9 fatty acid desaturase. J. Biol. Chem. 270, 29766–
29772.
20. Ausubel, F.M. & Frederick, M. (1991) Current Protocols in
Molecular Biology. Wiley, New York.
21. Bligh, E.G. & Dyer, W.J. (1959) A rapid method of total lipid
extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.
22. Yamamoto, K., Shibahara, A., Nakayama, T. & Kajimoto, G.
(1991) Determination of double-bond positions in methylene-
interrupted dienoic fatty acids by GC-MS as their dimethyl
disulfide adducts. Chem. Phys. Lipids 60, 39–50.
23. Schweizer, E. (1984) Genetics of fatty acid biosynthesis in yeast.
In Fatty Acid Metabolism and its Regulation (Numa, S., ed.), pp.
59–83. Elsevier Scientific Publishing Co, New York.
24. Paul, K.S., Jiang, D., Morita, Y.S. & Englund, P.T. (2001) Fatty
acid synthesis in African trypanosomes: a solution to the myristate
mystery. Trends Parasitol. 17, 381–387.
25. Roberts, C.W., McLeod, R., Rice, D.W., Ginger, M., Chance,
M.L. & Goad, L.J. (2003) Fatty acid and sterol metabolism:
potential antimicrobial targets in apicomplexan and trypanoso-
matid parasitic protozoa. Mol. Biochem. Parasitol. 126, 129–142.
26. de Lema, M.G. & Aeberhard, E.E. (1986) Desaturation of fatty
acids in Trypanosoma cruzi. Lipids 21, 718–720.
1086 G. A. Petrini et al.(Eur. J. Biochem. 271) Ó FEBS 2004