Desaturation of trans-octadecenoyl-acyl carrier protein by stearoyl-acyl carrier protein delta9 desaturase

Positional isomers of mono-unsaturated 18:1-ACP have been used as substrates for stearoyl-acyl carrier protein delta9 desaturase to test whether a C-H bond abstraction from either the C-9 or C-10 position could lead to rearranged products diagnostic for the production of an allylic radical intermediate. The reconstituted enzyme complex was able to desaturate trans-delta11-18:1-ACP and trans-delta7-18:1-ACP, but not trans-delta9-18:1-ACP, or any of the corresponding cis-isomers. Enzymatic desaturation of trans-delta11-18:1-ACP gave a single product, cis-delta9,trans-delta11-18:2-ACP, as characterized by gas chromatography-electron ionization mass spectrometry of the molecular ions, the fragmentation products of pyrrolidide and 4,4-dimethyloxazoline derivatives, and by comparison of chromatographic retention times with authentic standards. Reaction of trans-delta7-18:1-ACP gave two enzymic products, trans-delta7,cis-delta9-18:2 (approximately 80%) and trans-delta7,cis-delta11-18:2 (approximately 20%). The major product was likely formed in a reaction identical to that of 18:0-ACP desaturation, while the minor product was likely formed by alternative placement of the C-10 and C-11 positions of the substrate analog in a cis configuration relative to the diiron oxidant. Since none of the products observed are indicative of rearrangements originating with an allylic radical, a discussion of the origins and possible implications of these results is presented.     

Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex

Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from approximately 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to approximately 120 degrees in oxo-bridged diferric centers of the complex. Analysis of the shifts in nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta9D was used to investigate whether an O-atom from O(2) would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of approximately 70% yield of 18:1-ACP product in the presence of (18)O(2), no incorporation of an (18)O atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O(2) into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta9D catalysis are discussed.     

Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase

Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.     

大头金蝇酰基辅酶AΔ9去饱和酶cDNA克隆与原核表达

为深入研究大头金蝇Chrysomya megacephala (Fabricius)脂肪酸代谢关键功能基因酰基辅酶AΔ9去饱和酶(ACD9des),运用RT-PCR和RACE技术,获得其cDNA全长序列,并对其进行生物信息学分析。大头金蝇ACD9des基因cDNA (GenBank登录号为KF835695)全长1 429 bp,其中开放阅读框(ORF)为1 146 bp,编码381个氨基酸,5'UTR长度为138 bp,3'UTR约为114 bp。ORF编码的蛋白质分子量为43. 47 kD,等电点9. 06,氨基酸序列与其他昆虫酰基辅酶A去饱和酶一致性高达66%-93%,且含有由7个酰基辅酶A去饱和酶蛋白家族特有的保守模式(motif)所构成的指纹(IPR015876)。大头金蝇ACD9des在进化上与葱蝇Delia antiqua最趋于一致。将ACD9des的ORF克隆到原核表达载体p ET-44a(+),并利用Rosetta (DE3)感受态细胞进行ACD9des原核表达。Western Blot分析表明,IPTG诱导表达的特异性蛋白可以与anti-His抗体特异性结合,大小与预期理论值(43. 47 kDa)相符,为ACD9des。该蛋白主要存在于上清溶液中,为可溶性表达。最后利用含250 mM咪唑洗脱液和镍离子亲和层析柱对扩大培养获得的重组蛋白进行了纯化收集。本文的研究结果为大头金蝇功能基因的深入研究提供了坚实的基础。     

Expression and regulation of delta5-desaturase,delta6-desaturase,stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.     

Role of hydrophobic partitioning in substrate selectivity and turnover of the ricinus communis stearoyl acyl carrier protein delta(9) desaturase.

Stearoyl acyl carrier protein Delta(9) desaturase (Delta9D) uses a diiron center to catalyze the NADPH- and O(2)-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O(2) consumption and olefin production was found to be maximal for 18:0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the K(M) values were similar ( approximately 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by approximately 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K(M)) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K(M) values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The DeltaDeltaG(binding) estimated from the change in k(cat)/K(M) for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R. , and Tanford, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 289-293]. Since the K(M) values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta9D.     

Mineralocorticoids modify rat liver delta 6 desaturase activity and other parameters of lipid metabolism

The changes in linoleyl-CoA desaturase activity of rat liver microsomes were studied after a single intraperitoneal injection of 11-deoxycorticosterone or aldosterone at physiological doses. Fatty acid of plasma and different liver fractions, and physical properties of microsomal membranes were also investigated. It was found that the specific activity of delta 6 desaturase decreased 4-fold 24 hr after the injection of aldosterone or deoxycorticosterone. Pretreatment of the rats with aldosterone led to a significant decrease in the percent distribution of palmitic, arachidonic, docosapentaenoic and docosahexenoic acids, with a concomitant increase in palmitoleic, oleic and linoleic acids in plasma and liver homogenates, microsomes and cytosol fractions. A similar pattern was observed after deoxycorticosterone administration. The changes resulted in a decreased unsaturation index of all fractions studied and were well-correlated with the increase observed in fluorescence depolarization of the hydrophobic probe 1,6-diphenylhexatriene in liver microsomal membranes. The interlipid and lipid/protein ratios in microsomes remained constant after hormonal treatment. These results are consistent with the idea that the inhibition of delta 6 desaturase activity and the alterations in fatty acid composition induced by mineralocorticoids, are solely responsible for the measured decrease in liver microsomal membrane fluidity.     

Comparative expression in Escherichia coli of the native and hybrid genes for acyl-lipid delta(9) desaturase

Expression of the desC gene coding for acyl-lipid delta(9) desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid delta(9) desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acids.     

A desaturase-like protein from white spruce is a Delta(9) desaturase

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.     

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Δ¹¹ and Δ⁹ desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Δ⁹ desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.     

Modulation of delta 6 and delta 5 rat liver microsomal desaturase activities by dexamethasone-induced factor

This report supports evidence for the existence of a dexamethasone-induced factor that modulates fatty acid desaturase activities. Dexamethasone at a dose of 1 mg/rat produced a significant decrease in microsomal delta 6 and delta 5 desaturation activity 12 h after the injection. Both desaturase activities were depressed by a soluble factor present in the cytosolic fraction of cells, since the supernatant of microsomes separated at 110,000 X g from hormonal-treated rat liver homogenates, added to crude or washed control microsomes, was able to inhibit in vitro linoleic and homo-gamma-linolenic conversion to gamma-linolenic and arachidonic acids, respectively. The inhibitory factor was loosely bound to microsomes, since it was also present in a soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low-ionic-strength solution. Besides, trypsin digestion deactivates the dexamethasone-induced factor. Therefore, the depressing effect of glucocorticoids on delta 6 and delta 5 desaturation capacity depends on an unchanged protein structure present in the cytosolic fraction of the cell and whose biosynthesis is brought about by hormonal induction.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene

Strains of Saccharomyces cerevisiae bearing the ole1 mutation are defective in unsaturated fatty acid (UFA) synthesis and require UFAs for growth. A previously isolated yeast genomic fragment complementing the ole1 mutation has been sequenced and determined to encode the delta 9 fatty acid desaturase enzyme by comparison of primary amino acid sequence to the rat liver stearoyl-CoA desaturase. The OLE1 structural gene encodes a protein of 510 amino acids (251 hydrophobic) having an approximate molecular mass of 57.4 kDa. A 257-amino acid internal region of the yeast open reading frame aligns with and shows 36% identity and 60% similarity to the rat liver stearoyl-CoA desaturase protein. This comparison disclosed three short regions of high consecutive amino acid identity (greater than 70%) including one 11 of 12 perfect residue match. The predicted yeast enzyme contains at least four potential membrane-spanning regions and several shorter hydrophobic regions that align exactly with similar sequences in the rat liver protein. An ole1 gene-disrupted yeast strain was transformed with a yeast-rat chimeric gene consisting of the promoter region and N-terminal 27 codons of OLE1 fused to the rat desaturase coding sequence. Fusion gene transformants displayed near equivalent growth rates and modest lipid composition changes relative to wild type yeast control implying a significant conservation of delta 9 desaturase tertiary structure and efficient interaction between the rat desaturase and yeast cytochrome b5.     

Offspring metabolomic response to maternal protein restriction in a rat model of intrauterine growth restriction (IUGR)

Intrauterine growth restriction (IUGR), along with postnatal growth trajectory, is closely linked with metabolic diseases and obesity at adulthood. The present study reports the time-dependent metabolomic response of male offspring of rat dams exposed to maternal adequate protein diet during pregnancy and lactation (CC) or protein deprivation during pregnancy only (IUGR with rapid catch-up growth, RC) or through pregnancy and lactation (IUGR with slow postnatal growth, RR). Plasma LC-HRMS metabolomic fingerprints for 8 male rats per group, combined with multivariate statistical analysis (PLS-DA and HCA), were used to study the impact of IUGR and postnatal growth velocity on the offspring metabolism in early life (until weaning) and once they reached adulthood (8 months). Compared with CC rats, RR pups had clear-cut alterations in plasma metabolome during suckling, but none at adulthood; in contrast, in RC pups, alterations in metabolome were minimal in early life but more pronounced in the long run. In particular, our results pinpoint transient alterations in proline, arginine, and histidine in RR rats, compared to CC rats, and persistent differences in tyrosine and carnitine, compared to RC rats at adulthood. These findings suggest that the long-term deregulation in feeding behavior and fatty acid metabolism in IUGR rats depends on postnatal growth velocity.     

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.     

Blocking of the effect of delta1 tetrahydrocannabinol (delta1THC) on spontaneous motor activity in the rat by immunization with a protein conjugate of delta1THC

Rats actively immunized with porcine gamma globulin- hemisuccinate-Δ¹-tetrahydrocannabinol (PγG-HS-Δ¹THC) showed higher spontaneous motor activity after intraperitoneal administration of Δ¹THC at a dose of 10 mg. per kg. than did rats immunized with a control antigen, porcine gamma globulin-hemisuccinate-phenol (PγG-HS-Phenol). The capacity to neutralize the effect of Δ¹THC was found to depend on the degree of immunization; thus, the difference in mean spontaneous motor activity after injection of Δ¹THC was significant in rats which had received five injections of the immunogen over a period of 86 days, and not in those which had received only two injections over a period of 34 days.In view of the observations that Δ¹-tetrahydrocannabinol induces a decrease in spontaneous motor activity in rats, the observed neutralization of the effect of δ¹THC in animals receiving multiple injections of protein conjugates of Δ¹THC may be due to the binding of the drug by anti-THC antibodies (which are expected to be produced on active immunization with these conjugates), thus preventing Δ¹THC from reaching drug-receptor sites.     

Effects of (-)-trans- delta 9-tetrahydrocannabinol on serum prolactin in the pseudopregnant rat

Groups of pseudopregnant rats were injected intravenously with (-)-trans- delta 9-tetrahydrocannabinol (THC) to determine its effects on serum prolactin (PRL) and the maintenance of pseudopregnancy. A single injection of 4 mg THC/kg BW at 2400 h on the first day of leukocytic vaginal smears of pseudopregnancy (D-1) delayed the ensuing nocturnal PRL surge for approximately one hour. When THC (1.0 mg/kg BW) was administered hourly from 2400 h on D-1 through 1700 h on D-2, the nocturnal surge was blocked and serum PRL levels were suppressed until 0600 h on D-2, but not thereafter. Neither treatment altered the duration of pseudopregnancy. These results indicate that the nocturnal surge secretion of PRL during early pseudopregnancy in the rat is sensitive to THC suppression, but that this suppression is not adequate to influence the duration of pseudopregnancy. The mechanism through which THC exerts this action remains unknown.