Targeted mutagenesis of a fatty acid Delta6-desaturase from Mucor rouxii: role of amino acid residues adjacent to histidine-rich motif II

The amino acid residues serine at position 213 (S213) and lysine at position 218 (K218), which are present in close proximity to the histidine-rich motif II of Mucor rouxii fatty acid Delta(6)-desaturase isoform II, were targeted for studying structure-function relationships using site-directed mutagenesis. The mutants were functionally characterized in a heterologous host, Saccharomyces cerevisiae. Substrate specificity and preference studies revealed that S213 and K218 are involved in substrate recognition. K218 plays a role in substrate preference by involvement in the binding of substrates, particularly C15-C18 monoene fatty acids. Modification of the M. rouxii Delta(6)-desaturase therefore has potential in specifically altering substrate utilization for production of desired fatty acids.     

Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha

The Delta(6)-desaturase gene isoform II involved in the formation of gamma-linolenic acid (GLA) was identified from Mucor rouxii. To study the possibility of alteration of the synthetic pathway of essential fatty acids in the methylotrophic yeast, Hansenula polymorpha, the cloned gene of M. rouxii under the control of the methanol oxidase (MOX) promoter of H. polymorpha, was used for genetic modification of this yeast. Changes in flux through the n-3 and n-6 pathways in the transgenic yeast were observed. The proportion of GLA varied dramatically depending on the growth temperature and media composition. This can be explained by the effects of either substrate availability or enzymatic activity. In addition to the potential application for manipulating the fatty acid profile, this study provides an attractive model system of H. polymorpha for investigating the deviation of fatty acid metabolism in eukaryotes.     

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product ��8-desaturates 20:2n-6 and 20:3n-3

The mammalian Δ6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Δ8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Δ8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Δ6-desaturase activity is favored over Δ8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.     

Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase

Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.     

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.     

Differential expression of desaturases and changes in fatty acid composition during sporangiospore germination and development in Mucor rouxii

Polyunsaturated fatty acids (PUFAs), namely, oleic (C18:1), linoleic (C18:2), and gamma-linolenic acid (C18:3), constituted the majority in the total fatty acid content (44%) of sporangiospores of Mucor rouxii. At 30 degrees C, the germination begins within 1h at which time spore swelling occurs, followed by germ tube emergence within 3-4h. Throughout germination, an increase in gamma-linolenic acid (GLA) was observed and its content was highest at germ tube emergence. It took longer for sporangiospores of M. rouxii to germinate at sub-optimal temperatures (15 and 35 degrees C). However, the content of GLA was higher at the germ tube initiation than at the mycelial stage at all temperatures, suggesting the association of GLA and germination of sporangiospores. This finding was substantially confirmed by differential expression of delta9-, delta12-, and delta6-desaturase genes measured during spore germination. The expression of three desaturase genes parallels the pattern of GLA synthesis. By using RT-PCR techniques to follow gene expression, we found that mRNA of delta12- and delta6-desaturase genes were translated as soon as the spores were introduced into a fresh medium while the mRNA of delta9-desaturase gene could not be detected until 2h after introduction. A sharp increase in mRNA of delta6-desaturase genes correlated well with an increase in GLA content at germ tube emergence (4h). These results demonstrated that changes in fatty acid composition of sporangiospore of M. rouxii and differential expression of desaturase genes occurred during germination, and that extensive changes in GLA synthesis associated with some events in germination process.     

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.     

Molecular mechanisms underlying catalytic activity of delta 6 desaturase from Glossomastix chrysoplasta and Thalassiosira pseudonana

Delta 6 desaturase (FADS2) is a critical bifunctional enzyme required for PUFA biosynthesis. In some organisms, FADS2s have high substrate specificity, whereas in others, they have high catalytic activity. Previously, we analyzed the molecular mechanisms underlying high FADS2 substrate specificity; in this study, we assessed those underlying the high catalytic activity of FADS2s from Glossomastix chrysoplasta and Thalassiosira pseudonana. To understand the structural basis of this catalytic activity, GcFADS2 and TpFADS2 sequences were divided into nine sections, and a domain-swapping approach was applied to examine the role of each section in facilitating the catalytic activity of the overall protein. The results revealed two regions essential to this process: one that extends from the end of the fourth to the beginning of the fifth cytoplasmic transmembrane domain, and another that includes the C-terminal region that occurs after the sixth cytoplasmic transmembrane domain. Based on the domain-swapping analyses, the amino acid residues at ten sites were identified to differ between the GcFADS2 and TpFADS2 sequences, and therefore further analyzed by site-directed mutagenesis. T302V, S322A, Y375F, and M384S/M385 substitutions in TpFADS2 significantly affected FADS2 catalytic efficiency. This study offers a solid basis for in-depth understanding of catalytic efficiency of FADS2.     

Isolation and Characterization of Δ6-Desaturase,an ELO-Like Enzyme and Δ5-Desaturase from the Liverwort Marchantia Polymorpha and Production of Arachidonic and Eicosapentaenoic Acids in the Methylotrophic Yeast Pichia Pastoris

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.     

A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.     

Ethanol-induced changes in expression profiles of cell growth, fatty acid and desaturase genes of Mucor rouxii

We investigated the response of Mucor rouxii to ethanolic stress conditions. A differential response was found that was dependent on growth phase and ethanol concentration. 5% (v/v) ethanol showed an inhibitory effect on the mycelial growth of all stages. However, the ethanol sensitivity was specifically observed in active growing phases (12 and 21 h-grown cultures), in which the biomass and ratio of unsaturated/saturated fatty acids (UFA/SFA) decreased greatly after ethanol exposure compared to non-ethanol adding culture. With respect to different ethanol concentrations, M. rouxii was tolerant to low ethanol concentrations (about 1-3%, v/v) such that there was not much change in biomass and UFA/SFA ratio, in contrast to the 5% ethanol-added culture. We also showed the molecular basis of this response mechanism, demonstrating that expression of Delta(9)-, Delta(12)- and Delta(6)-desaturase genes, responsible for fatty acid desaturation in M. rouxii, were coordinately down-regulated upon exposure to ethanol stress.     

Competition between 24:5n-3 and ALA for Delta 6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.     

Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.     

少根根霉△6-脂肪酸脱氢酶基因在毕赤酵母中的表达

△6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶.在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码△6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的△6-脂肪酸脱氢酶基因.把少根根霉△6-脂肪酸脱氢酶基因(RAD6)亚克隆到表达载体pPIC3.5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达.提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16:1、C17:1、C18:1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性.此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平.综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析△6-脂肪酸脱氢酶基因的功能.     

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.     

Genetic manipulation of gamma-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.)

A robust Agrobacterium-mediated transformation procedure was developed for Rigel, a commercial cultivar of evening primrose, and used to deliver a cDNA encoding a Delta(6)-desaturase from borage under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the transformed plants demonstrated an altered profile of polyunsaturated fatty acids, with an increase in gamma-linolenic acid and octadecatetraenoic acid in leaf tissues when compared with control lines.