Identification of a fatty acid Delta11-desaturase from the microalga Thalassiosira pseudonana

A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana. Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e. a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain. Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Delta11. To our knowledge, until this report, Delta11-desaturase activity had only been detected in insect cells.     

Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

A second functional delta5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum.

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www. csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Delta5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino-acid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Delta5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing Delta5 fatty acid desaturase. As we have already reported one other Delta5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Delta5 fatty acid desaturase genes. The substrate specificities of the two functional Delta5 desaturases of D. discoideum were also examined.     

Trypanosoma brucei oleate desaturase may use a cytochrome b5-like domain in another desaturase as an electron donor.

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 Arabidopsis thaliana Delta12 desaturase, respectively, and 51% with A. thaliana omega3 desaturases. The histidine tracks that compose the iron-binding active centers of the enzyme were more similar to those of the omega3 desaturases. Expression of the trypanosome gene in Saccharomyces cerevisiae resulted in the production of fatty acids that are normally not synthesized in yeast, namely linoleic acid (18:2Delta9,12) and hexadecadienoic acid (16:2Delta9,12), 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 supplemented with 19:1Delta10 fatty acid yielded 19:2Delta10,13, indicating 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-unsaturated fatty acid production, although it was not totally abolished. Based on the regioselectivity and substrate preferences, we characterized the trypanosome enzyme as a cytochrome b5-dependent oleate desaturase. Expression of the ORF in a double mutant (ole1Delta,cytb5Delta) abolished all 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 cytb5Delta single mutant.     

Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA delta 9-desaturase of the cabbage looper moth, Trichoplusia ni.

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea

Two cDNAs with sequence similarity to fatty acid desaturase genes were isolated from the phytopathogenic fungus, Claviceps purpurea. The predicted amino acid sequences of the corresponding genes, named CpDes12 and CpDesX, share 87% identity. Phylogenetic analysis indicates that CpDes12 and CpDesX arose by gene duplication of an ancestral Delta(12)-desaturase gene after the divergence of Nectriaceae and Clavicipitaceae. Functional expression of CpDes12 and CpDesX in yeast (Saccharomyces cerevisiae) indicated that CpDes12 is primarily a "Delta(12)"-desaturase, whereas CpDesX is a novel desaturase catalyzing "Delta(12)," "Delta(15)," and "omega(3)" types of desaturation with omega(3) activity predominating. CpDesX sequentially desaturates both 16:1-9c and 18:1-9c to give 16:3-9c,12c,15c and 18:3-9c,12c,15c, respectively. In addition, it could also act as an omega(3)-desaturase converting omega(6)-polyunsaturates 18:3-6c,9c,12c, 20:3-8c,11c,14c, and 20:4-5c,8c,11c,14c to their omega(3) counterparts 18:4-6c,9c,12c,15c, 20:4-8c,11c,14c,17c, and 20:5-5c,8c,11c,14c,17c, respectively. By using reciprocal site-directed mutagenesis, we demonstrated that two residues (isoleucine at 152 and alanine at 206) are critical in defining the catalytic specificity of these enzymes and the C-terminal amino acid sequence (residues 302-477) was also found to be important. These data provide insights into the nature of regioselectivity in membrane-bound fatty acid desaturases and the relevant structural determinants. The authors suggest that the regios-electivity of such enzymes may be best understood by considering the relative importance of more than one regioselective preference. In this view, CpDesX is designated as anu + 3(omega(3)) desaturase, which primarily references an existing double bond (nu + 3 regioselectivity) and secondarily shows preference for omega(3) desaturation.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.

Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.     

Delta11 desaturases of Trichoplusia ni and Spodoptera littoralis exhibit dual catalytic behaviour

The Delta(11) desaturases found in moths such as Spodoptera littoralis play a critical role in the biosynthesis of their sex pheromones. The ability to functionally express these enzymes in yeast has allowed one to study the transformation of long-chain fatty acyl substrates to their 11-ene products in greater mechanistic detail. In this article, we report on the detection and quantitation of a minor 11-hydroxylated byproduct (0.1% of total fatty acids), which is formed by the Delta(11) desaturases found in Trichoplusia ni and Spodoptera littoralis. The position of the hydroxyl group was determined by characteristic mass spectral fragmentation of the trimethylsilyl derivatives and is in accord with predictions based on previous mechanistic investigations of the Spodoptera Delta(11) desaturase. The level of 11-hydroxylation was insensitive to the mode of desaturase expression (constitutive vs. induced) and the presence or absence of a b5-fusion domain. Our findings suggest that in future, a search for hydroxylated products should be included in functional analyses of insect desaturase genes.     

A small membrane-peripheral region close to the active center determines regioselectivity of membrane-bound fatty acid desaturases from Aspergillus nidulans

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.     

The phylogenetic relationship of possible progenitors of the cultivated peanut

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid composed of A and B genomes. The phylogenetic relationship among the cultivated peanut, wild diploid, and tetraploid species in the section Arachis was studied based on sequence comparison of stearoyl-ACP desaturase and oleoyl-PC desaturase. The topology of the trees for both fatty acid desaturases displayed two clusters; one cluster with A genome diploid species and the other with B genome diploid species. The two homeologous genes obtained for each of the two fatty acid desaturases from the tetraploid species A. hypogaea and A. monticola were separated into the A and B genome clusters, respectively. The gene phylogenetic trees showed that A. hypogaea is more closely related to the diploid species A. duranensis and A. ipaensis than to the wild tetraploid species A. monticola, suggesting that A. monticola is not a progenitor of the cultivated peanut. In addition, for the stearoyl-ACP desaturase, the A. duranensis sequence was identical with one of the sequences of A. hypogaea and the A. ipaensis sequence was identical with the other. These results support the hypothesis that A. duranensis and A. ipaensis are the most likely diploid progenitors of the cultivated tetraploid A. hypogaea.     

Membrane topology of the acyl-lipid desaturase from Bacillus subtilis

The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.     

Structural and functional conservation and divergence among acyl-CoA desaturases of two noctuid species, the corn earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni

In this report, we describe the structural and functional analyses of four acyl-CoA desaturase-encoding cDNAs that we isolated from RNA expressed in the pheromone gland of the corn earworm, Helicoverpa zea. We deduced the homology relationships of the encoded proteins, designated HzPGDs1, HzPGDs2, HzPGDs3 and HzFBDs, to each other and to previously described desaturases of the cabbage looper moth, Trichoplusia ni, the fly, Drosophila melanogaster, and other more distantly related organisms. We also isolated genomic DNA fragments of the four H. zea desaturase-encoding genes, determined the locations of introns present in them, and compared them to conserved intron positions in reported desaturase genes of other species. We measured the levels of the four desaturase mRNAs in H. zea pheromone glands and larval fat bodies by RT-PCR. We established the functional identities of the deduced proteins HzPGDs1 and HzPGDs2, encoded by the two desaturase mRNAs that are differentially and abundantly expressed in pheromone glands of sexually mature adult H. zea females, by functional expression of their encoding cDNAs in a desaturase-deficient mutant, ole1, of the yeast Saccharomyces cerevisiae. We compared the unique unsaturated fatty acid profiles of HzPGDs1- and HzPGDs2-expressing transformants to those of strains expressing previously described Delta11 and Delta9 desaturases of T. ni.