深黄被孢霉△~6-脂肪酸脱氢酶基因的克隆及序列分析

γ－亚麻酸（ＧＬＡ,Ｃ１８：３△６,９,１２）是由△６－脂肪酸脱氢酶以亚油酸（ＬＡ,Ｃ１８：２△９,１２）为底物,在Ｃ６位脱氢形成的。由于在人体中,γ－亚麻酸是花生四烯酸、前列腺素类和白三烯类等生理活性物质的前体物,而深黄被孢霉是目前用于微生物发酵生产γ－亚麻酸的主要菌株。本文根据脂肪酸脱氢酶的保守区设计引物,利用反转录聚合酶链式反应从丝状真菌深黄被孢霉中克隆了编码△６－脂肪酸脱氢酶的ｃＤＮＡ,全长为１３７４个核苷酸,编码４５７ 个氨基酸,但与其他位点的脂肪酸脱氢酶不同的是, △６－脂肪酸脱氢酶在其序列的 Ｎ 端特有细胞色素 ｂ５（Ｃｙｔｂ５）区。这是国际上对深黄被孢霉△６－脂肪酸脱氢酶基因的首次报道。     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Tonoplast subcellular localization of maize cytochrome b5 reductases

Plant cytochrome b5 reductases (b5R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem. J. 338, 499-505). Maize b5R isoforms are produced by a small multi-gene family. The NFR cDNAs were shown to encode active b5Rs by heterologous expression in yeast. Both reductases, in addition to Fe3+-chelates, efficiently reduced Cu2+-chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b5Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast-enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b5R. This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b5Rs may have previously unexpected functions.     

The Effect of Dietary Sterol on the Activity of Fatty Acid Desaturases Isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a Δ9, a Δ12 and a Δ6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to ?-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the Δ6 and Δ12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

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.     

A possible cyclic AMP-mediated regulation of microsomal fatty acyl-CoA desaturation system in Tetrahymena microsomes

Profound alterations in the microsomal fatty acyl-CoA desaturase activities and cyclic AMP production of a unicellular eukaryote, Tetrahymena pyriformis NT-1, originally grown in the glucose-deficient medium, were observed, following the administration of glucose or beta-adrenergic agonists such as epinephrine and isoproterenol. There was a great increase of stearoyl-CoA (delta 8) desaturase activity coincident with a 2-fold decrease of oleoyl-CoA (delta 12) desaturase activity over the first 2 h after administration of these compounds. During this period of time, it was found that the production in vivo of labeled oleic acid from [14C]acetic or [3H]palmitic acid increases 2-fold and the formation in vivo of each labeled linoleic and gamma-linolenic acids drastically decreases. Glucose or beta-adrenergic agonists caused an increase of stearoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b5 but depressed oleoyl-CoA-stimulated reoxidation rate of b5, indicating that both desaturase activities are controlled by the respective terminal components of the desaturase system. A significant and reproducible increase of adenylate cyclase activity and a slight decrease of cyclic AMP phosphodiesterase activity were observed to occur within the first 2 h after the addition of these compounds, when cyclic AMP content in Tetrahymena cell rose by 3-4-fold. Propranolol, a beta-adrenergic blocker, abolished the effects of glucose or beta-adrenergic agonists on the activities of fatty acyl-CoA desaturases and the terminal components as well as cyclic AMP production of cells. These results suggest that glucose and beta-adrenergic agonists may modulate the microsomal fatty acyl-CoA desaturase system in Tetrahymena by acting through the increase of intracellular cyclic AMP content.     

Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis

In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.     

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.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

Effect of clofibrate on fatty acid desaturation of rats treated with ethanol

The effect of clofibrate and ethanol in the rat was studied on the following aspects of lipid composition and metabolism: liver delta 5, delta 6 and delta 9 fatty acid desaturases, fatty acid synthetase and fatty acid desaturase microsomal electron transport chain activity and serum cholesterol, triacylglycerols and high (HDL), low (LDL) and very low density lipoprotein (VLDL) levels. Clofibrate administered for 9 days (0.3% W/W) did not modify the relative composition of liver phospholipids and cholesterol, but did diminish triacylglycerol levels increased by ethanol. This effect could be explained by the possible beta-adrenergic blocking properties of clofibrate or by an increased activity of peroxisomal beta-oxidation. Clofibrate also promoted a decrease in serum cholesterol and triacylglycerol levels, delta 6 desaturase activity and a suppression of the electron transport chain as measured by NADH cytochrome b5 reductase and NADH cytochrome c reductase. The drug increased delta 9 desaturase activity and fatty acid synthetase, while no effect could be found in delta 5 desaturase activity. The hypocholesterolenic effect of clofibrate can not be explained through the delta 6 desaturase inhibition, or the fatty acid synthetase enhancement. Ethanol increased the HDL and VLDL and lowered LDL serum concentrations, while clofibrate reversed these results. Considering that clofibrate could have antiatherosclerotic effect in the rat, it is difficult to explain it through these changes in lipoprotein levels, since according to Miller and Miller low HDL levels are predictive of coronary heart disease.     

Temporal and spatial gene expression of cytochrome B5 during flower and fruit development in olives

We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4–6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 °C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.     

Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from <Emphasis Type="Bold">α</Emphasis>-Linolenic Acid in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Fish are the only major dietary source for humans of -3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b₅ domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an -3 5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from -linolenic acid (18:3n-3). The desaturase showed only low levels of 6 activity toward C₁₈ polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C₁₈ > C₂₀ > C₂₂. Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.     

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.     

Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by 13C labelling and desaturase inhibitor experiments

The lipids of the heterotrophic microalga Crypthecodinium cohnii contain the omega-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (22:6) to a level of over 30%. The pathway of 22:6 synthesis in C. cohnii is unknown. The ability of C. cohnii to use 13C-labelled externally supplied precursor molecules for 22:6 biosynthesis was tested by 13C NMR analysis. Furthermore, the presence of desaturases (typical for aerobic PUFA synthesis) was studied by the addition of specific desaturase inhibitors in the growth medium. The addition of 1-(13)C acetate or 1-(13)C butyrate in the growth medium resulted in 22:6 with only the odd carbon atoms enriched. Apparently, two-carbon units were used as building blocks for 22:6 synthesis and butyrate was first split into two-carbon units prior to incorporation in 22:6. When 1-(13)C oleic acid was added to the growth medium, 1-(13)C oleic acid was incorporated into the lipids of C. cohnii but was not used as a precursor for the synthesis of 22:6. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation in C. cohnii. The fatty acid profile, however, was not altered. In contrast, in the arachidonic acid-producing fungus, Mortierella alpina, these inhibitors not only decreased the lipid content but also altered the fatty acid profile. Our results can be explained by the presence of three tightly regulated separate systems for the fatty acid production by C. cohnii, namely for (1). the biosynthesis of saturated fatty acids, (2). the conversion of saturated fatty acids to monounsaturated fatty acids and (3). the de novo synthesis of 22:6 with desaturases involved.