Stearoyl-acyl Carrier Protein Desaturase Research Articles

Azide and Acetate Complexes Plus Two Iron-depleted Crystal Structures of the Di-iron Enzyme Δ9 Stearoyl-Acyl Carrier Protein Desaturase: IMPLICATIONS FOR OXYGEN ACTIVATION AND CATALYTIC INTERMEDIATES

Delta9 stearoyl-acyl carrier protein (ACP) desaturase is a mu-oxo-bridged di-iron enzyme, which belongs to the structural class I of large helix bundle proteins and that catalyzes the NADPH and O2-dependent formation of a cis-double bond in stearoyl-ACP. The crystal structures of complexes with azide and acetate, respectively, as well as the apoand single-iron forms of Delta9 stearoyl-ACP desaturase from Ricinus communis have been determined. In the azide complex, the ligand forms a mu-1,3-bridge between the two iron ions in the active site, replacing a loosely bound water molecule. The structure of the acetate complex is similar, with acetate bridging the di-iron center in the same orientation with respect to the di-iron center. However, in this complex, the iron ligand Glu196 has changed its coordination mode from bidentate to monodentate, the first crystallographic observation of a carboxylate shift in Delta9 stearoyl-ACP desaturase. The two complexes are proposed to mimic a mu-1,2 peroxo intermediate present during catalytic turnover. There are striking structural similarities between the di-iron center in the Delta9 stearoyl-ACP desaturase-azide complex and in the reduced rubrerythrin-azide complex. This suggests that Delta9 stearoyl-ACP desaturase might catalyze the formation of water from exogenous hydrogen peroxide at a low rate. From the similarity in iron center structure, we propose that the mu-oxo-bridge in oxidized desaturase is bound to the di-iron center as in rubrerythrin and not as reported for the R2 subunit of ribonucleotide reductase and the hydroxylase subunit of methane monooxygenase. The crystal structure of the one-iron depleted desaturase species demonstrates that the affinities for the two iron ions comprising the di-iron center are not equivalent, Fe1 being the higher affinity site and Fe2 being the lower affinity site.

Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase.

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.

Cloning and expression of fatty acids biosynthesis key enzymes from sunflower ( Helianthus annuus L.) in Escherichia coli

To further characterize the stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) and the acyl-ACP thioesterase FatB (EC 3.1.2.14) activities from sunflower seeds, we cloned, sequenced and expressed the recombinant genes in Escherichia coli. We obtained two partially purified proteins, His-SAD and His-FATB, each of about 45 000 Da. The expression of either proteins produced changes in the E. coli fatty acid profile indicating the functionality of the recombinant proteins. While the expression of His-SAD produced an effect similar to that produced by overexpression of the fabA gene, responsible for the fatty acid desaturation in E. coli, the expression of His-FATB gave rise to an unbalance between unsaturated fatty acids and a toxic effect in E. coli.

Identification of new nodulin cDNAs from yellow lupine by differential display

In this study several sequences that represent yellow lupine nodulin genes are identified. Four different cDNA clones were isolated from a lupine nodule cDNA library and sequenced. Database search revealed that the deduced amino acid of one clone, DD3A20, shows near 80% amino acid similarity to plant stearoyl-acyl carrier protein desaturases and contains crucial amino acids in the fatty acid binding domain which are characteristic for stearoyl-ACP desaturases. The other sequences seem to be unique. Nearly identical signal peptide sequences are present at the N-termini of the DD4A9 and DD2T15 putative proteins. Northern analysis indicated that the pattern of expression of the Lldd4A9, Lldd2A18 and Lldd2T15 genes is the same as that of the lupine leghemoglobin gene LllbII, while the desaturase gene Ll3A20 is turned on at least 3 days earlier. Ll3A20 is also expressed in lupine fruits. RT-PCR analysis of the Lldd3A20 and Lldd2T15 expression confirmed organ-specific and temporal pattern of expression in nodules.

Staphylococcus aureus nuclease is a useful secretion reporter for mycobacteria.

A secretion reporter system based on Staphylococcus aureus nuclease (nuc) was developed for use in mycobacteria. Fusion of secretion signals to the reporter cloned in a shuttle vector, pBPnuc1, resulted in halo formation around colonies of Mycobacterium smegmatis and Mycobacterium tuberculosis grown on DNase agar plates containing Methyl Green indicator dye. This in-situ detection system was used to identify secreted proteins by screening a pBPnuc1::H37Rv nuc gene fusion library in M. smegmatis. The clones identified in this screen all formed colony halos when present in M. tuberculosis grown on indicator media. The proteins corresponded to DesA2, a stearoyl-acyl carrier protein desaturase, PepA, a putative serine protease and the Apa antigen, which is the ATP-binding subunit of an ABC transport system. Of these proteins, only PepA and Apa contained recognizable leader peptides.

Isolation and Characterization of Two Promoters from Linseed for Genetic Engineering

ABSTRACTLinseed (Linum usitatissimum L.) is an important oilseed crop worldwide and is cultivated for the high level of linolenic acid (18:3) in its seed oil. Currently, there is a concerted effort to improve linseed by genetic engineering. This will require appropriate transgenes and tissue‐specific or constitutive promoters. We report the isolation and characterization of two linseed promoters from a two‐member gene family encoding the enzyme stearoyl‐acyl carrier protein desaturase (SAD). The SAD1 and SAD2 gene promoter were each fused transcriptionally with the reporter gene for β‐glucuronidase (uidA; GUS) and were transferred to linseed to study their expression pattern. In transgenic linseed, GUS activity mediated by the SAD2 promoter appeared to be constitutive and was detected in leaves, apices, stem, roots, flower buds, flowers, and seeds. In contrast, GUS activity mediated by the SAD1 promoter appeared to be root‐ and seed‐specific. In developing seeds, both the promoters exhibited a temporal expression pattern concomitant with protein and lipid biosyntheses. The GUS activity could be detected as early as 4 days after pollination (dap) and in mature seeds (∼50 dap) with the highest activities around mid‐development. The first pair of linseed promoters will be useful for manipulating the expression of indigenous as well as transgenes in linseed to create value‐added cultivars.

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

The Crystal Structure of an Azide Complex of the Diferrous R2 Subunit of Ribonucleotide Reductase Displays a Novel Carboxylate Shift with Important Mechanistic Implications for Diiron-Catalyzed Oxygen Activation

The dinuclear Fe-center in the R2 protein of ribonucleotide reductase catalyzes oxygen activation chemistry leading to generation of the essential stable tyrosyl radical. Related oxygen reactions occur in several other diiron-containing enzyme systems where highly oxidative reaction intermediates are required to activate the substrates. Two such examples are methane monooxygenase and Δ9 stearoyl-acyl carrier protein desaturase where oxygen activation takes place at Fe-centers whose structures are similar to the Fe-center in R2. In an attempt to structurally characterize the nature of the dioxygen cleavage reaction performed by these proteins we have determined the crystal structures of two different forms of the diferrous R2 protein in the presence of azide, a potential Fe ligand. In crystals of the wt protein no azide binding was detected. The mutant protein F208A/Y122F has a larger hydrophobic pocket around the Fe-center and in the structure of this protein azide bind as an η1-terminal ligand to Fe2, the Fe ion farthest away from the tyrosine residue to be oxidized in the radical generation reaction. Glu 238, the Fe ligand most exposed into the hydrophobic pocket, coordinates the Fe-center in a novel μ-(η2,η1) bridging mode with one of the carboxylate oxygen atoms forming a bridge between the two iron ions and the other oxygen being coordinated to Fe2. Through this bridging the Fe−Fe distance is shortened to about 3.4 Å as compared to 3.9 Å for the structure of the reduced wt protein. On the basis of the novel carboxylate shift and recent data on the spectroscopic properties of the key intermediate X we propose a unique structure for intermediate X and a detailed mechanism for dioxygen cleavage. This mechanism suggests an asymmetric oxygen cleavage with a terminal oxo/hydroxo group as the major species responsible for substrate activation.

Ethylene enhances the antifungal lipid content in idioblasts from avocado mesocarp

It has previously been demonstrated that exposure of whole avocado fruits cv. Fuerte to 40 μl/l ethylene for 3 h enhances the level of antifungal 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (diene) in the fruit pericarp transiently, without affecting disease resistance to C. gloeosporioides. Exposure of 1–2 mm slices of fruit pericarp and mesocarp to ethylene enhanced the level of the antifungal diene in the mesocarp only. Since most of the antifungal diene in the mesocarp is compartmentalized in idioblasts the effect of ethylene was tested on isolated idioblasts. Exposure of idioblasts to ethylene increased the level of antifungal diene twofold within 3 hs. This effect was temperature dependent. Three hours exposure of idioblasts to ethylene at 35°C doubled the diene content compared to less than 50% increase after three hours at 20°C. Incubation of idioblast with [2-14C] malonyl- CoA or [1-14C] acetate in the presence of ethylene, showed the incorporation of the label into a compound that co-chromatographed with the antifungal diene. NMR identified the compound induced by ethylene and released from the cells as the antifungal diene. cDNA libraries were constructed from mesocarp tissue of ethylene treated and untreated fruits. Using the castor stearoyl-acyl carrier protein (ACP) desaturase gene, a putative stearoyl (ACP) desaturase clone of avocado, was detected. Transcripts of this clone were 2.5 fold higher in the ethylene treated than in the untreated tissue. The present report will discuss the possibility that ethylene can induce the synthesis of the antifungal diene in idioblasts of avocado fruits.

Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.

DFLP, SSCP, and SSR markers for Δ9-stearoyl-acyl carrier protein desaturases strongly expressed in developing seeds of sunflower: intron lengths are polymorphic among elite inbred lines

Stearoyl-acyl carrier protein desaturase (SAD, EC 1.14.99.6) produces oleic acid (18:1 Δ9) by desaturating 18:0. SAD genes have been targets for breeding and engineering oilseed crops with increased stearic acid (18:0). Our aim was to clone, describe, and develop genetic markers for the SAD genes of sunflower (Helianthus annuus L.). Nineteen SAD cDNA clones were partially sequenced and found to belong to two groups. Full-length cDNAs from each group (SAD6 and SAD17) were completely sequenced. The amino acid identity of SAD6 and SAD17 was 89%. Both genes were strongly expressed in developing seeds, moderately expressed in leaves and flowers, and weakly expressed in cotyledons, roots, and stems. One intron was found in SAD6 and two introns were found in SAD17. The SAD introns from two inbred lines (HA370 and HA372) were sequenced and found to vary in length and nucleotide sequence. The length variants were caused by monomeric repeat length differences, insertions, and deletions. Three long poly-T repeats (T9 to T39) were found in one of the SAD17 introns. Three short adjacent CA repeats were found in the 5′-untranslated region of SAD6. DNA fragment length polymorphism (DFLP), single-strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for SAD6 and SAD17 by developing primers to flank introns or the CA repeats. Two of six DFLP, four of six SSCP, and one of two SSR markers were polymorphic among eight elite inbred lines. The polymorphic information contents for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Most of the polymorphisms were caused by intron fragment length polymorphisms. Introns may be an excellent source of hypervariable markers in sunflower and other crop plants.

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.

Structure and Expression of Two Seed-Specific cDNA Clones Encoding Stearoyl-Acyl Carrier Protein Desaturase from Sesame, Sesamum indicum L.

We have isolated two cDNA clones (CDES01) and 04) encoding stearoyl-acyl carrier protein desaturase (SACPD; EC 1.14.99.6) from immature sesame seeds, and have analyzed accumulation levels of the corresponding mRNAs at different stages and organs in sesame. Clone CDES01 contains an open reading frame coding for a 396-amino acid protein of 45 kDa. CDES04 encodes a partial sequence of 141-amino acids. Deduced amino acid sequences of both clones exhibit a high identity to those of other plant SACPD cDNAs. Northern blots probed with CDES01 and CDES04 indicate that both messages accumulate in a seed-specific manner with a peak at 21 days after anthesis. However, expression patterns also indicate that regulation between CDES01 and CDES04 are slightly different. The CDES01 message accumulates at a low level in young leaves in addition to seeds, whereas accumulation of the RNA transcript corresponding to CDES04 is restricted to seeds. This observation implies the presence of at least two isozymes of SACPD having overlapping but slightly distinct functions in sesame.

Molecular Cloning of the Gene Encoding Stearoyl-Acyl Carrier Protein Desaturase in Brassica juncea

Metabolic engineering of the pathways of lipid biosynthesis has generated transgenic oilseed crops with enhanced levels of specialty fatty acids of Industrial value. Stearic acid, a 18:0 saturated fatty acid, is one such important fatty acid. Stearoylacyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis and converts stearoyl-ACP to oleoyl-ACP. We have cloned the complete coding region of the gene for this enzyme in Brassica juncea. Based on the sequence information of the gene in B. napus, 27-mer forward and reverse primers were designed each of which incorporated a Sal I restriciton site at the end. The primers were used to fish out the desaturase gene from B. juncea genome by polymerase chain reaction (PCR). The PCR product conformed to the average size of the coding region of the gene in B. napus. The PCR product was cloned in the pGem-T vector. The cloning was reconfirmed by restriction enzyme analysis and by PCR of the recombinant plasmid. The potential use of this gene in molecular farming of designer oilseed brassicas is discussed.

Nucleotide Sequence of a Stearoyl-Acyl Carrier Protein Desaturase cDNA from Developing Seeds of Rice

Nucleotide Sequence of a Stearoyl-Acyl Carrier Protein Desaturase cDNA from Developing Seeds of Rice

A Mutant of Arabidopsis with Increased Levels of Stearic Acid

A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol.

Changes in fatty-acid composition and stearoyl-acyl carrier protein desaturase expression in developing Theobroma cacao L. embryos

The fatty-acid composition and expression of stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) were studied in developing Theobroma cacao L. embryos between 95 and 145 days after pollination (DAP). During this interval, the fraction of lipid contributed by linoleic and palmitic acids decreased, and the fraction contributed by stearic and oleic acids increased. After 115 DAP, stearic, palmitic, and oleic acids accumulated at nearly the same rate, and the fatty-acid composition resembled that of cocoa butter. The ratio of C-18 unsaturated fatty acids to stearic acid decreased between 95 and 115 DAP, then leveled-off. Thus, lipid composition changed from membrane-like to cocoa-butter-like in only 20 d out of the 170 d required for embryos to reach maturity. Levels of stearoyl-ACP desaturase were determined by Western blot analyses using antibodies to avocado stearoyl-ACP desaturase. Western blots revealed a single 38-kDa band — corresponding to the expected size of the stearoyl-ACP-desaturase monomer — in extracts from 95- and 105-DAP embryos. Although the amount of stearoyl-ACP-desaturase protein was 2.3-fold higher at 95 than at 105 DAP when equal amounts of protein were loaded per lane, no difference in the abundance of stearoyl-ACP desaturase was observed when the protein loaded per lane was standardized for equal amounts of DNA.

Palmitoleate formation by soybean stearoyl-acyl carrier protein desaturase

Palmitoyl (hexadecanoyl)-acyl carrier protein (ACP) was found to be an alternate substrate for recombinant soybean stearoyl (octadecanoyl)-ACP Δ 9-clesaturase. The fatty acid product was identified as palmitoleic acid (( Z)-hexadec-9-enoic acid), whic suggests that the locus of desaturation was fixed with respect to the carboxyl, not methyl, end of each substrate acyl group. The apparent specificity factor ( V max/ K m) of the desaturase for stearoyl-ACP was > 100-fold larger than for palmitoyl-ACP, mostly because of the difference in V max for the two substrates.

The Primary Structure of a cDNA Clone of the Stearoyl-Acyl Carrier Protein Desaturase Gene from Potato (Solanum tuberosum L.)

The Primary Structure of a cDNA Clone of the Stearoyl-Acyl Carrier Protein Desaturase Gene from Potato (<i>Solanum tuberosum</i> L.)

Preliminary crystallographic data for stearoyl-acyl carrier protein desaturase from castor seed

Recombinant stearoyl-acyl carrier protein desaturase (EC 1.14.99.6) from castor seed has been crystallized with polyethylene glycol 8000 as precipitant. The crystals are orthorhombic, space group P2 12 12 1 with cell dimensions a = 81.3, b = 146.4 and c = 197.7 A ̊ . The observed diffraction pattern extends to at least 2.5 Å resolution. Rotation function calculations indicate a non-crystallographic 3-fold rotation axis parallel to the crystallographic a-axis. Perpendicular to this axis, 2-fold rotation axes were found at 30 ° intervals, i.e. maxima at κ = 180 °, φ = 90 ° and ω = 30 ° and 60 °, respectively. Together with the packing density of the crystals ( V m = 2.4 Å) 3/Da for n = 6), these results suggest, that the crystal asymmetric unit most likely contains a hexamer of desaturase subunits.