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: toniuttaro@yahoo.com.ar 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 (http://tigrblast.tigr.org). 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 http://www.ch.embnet.org/software/TMPRED_ 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. 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