The crystal structure of the ivy Delta4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the Delta(4) desaturation of 16:0-ACP and the Delta(9) desaturation of 18:0-ACP and further desaturates Delta(9)-16:1 or Delta(9)-18:1 to the corresponding Delta(4,9) dienes. The crystal structure of the enzyme has been solved to 1.95 A resolution, and both the iron-iron distance of approximately 3.2A and the presence of a mu-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His(227) coordination to Fe2 is lost, and the side chain of Glu(224), which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

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.     

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.     

Desaturation,chain scission,and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.     

Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed

A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1⁹)* and cis-vaccenic (18:1¹¹) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed ⁹ desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known ⁹-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized ⁹-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a⁹ -18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.     

Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein.

We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1. Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix. Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields. Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled. ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B. Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient. The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports.     

Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase

Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.     

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.     

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.     

Properties of two multifunctional plant fatty acid acetylenase/desaturase enzymes.

The properties of the Delta6 desaturase/acetylenase from the moss Ceratodon purpureus and the Delta12 acetylenase from the dicot Crepis alpina were studied by expressing the encoding genes in Arabidopsis thaliana and Saccharomyces cerevisiae. The acetylenase from C. alpinaDelta12 desaturated both oleate and linoleate with about equal efficiency. The desaturation of oleate gave rise to 9(Z),12(E)- and 9(Z),12(Z)-octadecadienoates in a ratio of approximately 3 : 1. Experiments using stereospecifically deuterated oleates showed that the pro-R hydrogen atoms were removed from C-12 and C-13 in the introduction of the 12(Z) double bond, whereas the pro-R and pro-S hydrogen atoms were removed from these carbons during the formation of the 12(E) double bond. The results suggested that the Delta12 acetylenase could accommodate oleate having either a cisoid or transoid conformation of the C(12)-C(13) single bond, and that these conformers served as precursors of the 12(Z) and 12(E) double bonds, respectively. However, only the 9(Z),12(Z)-octadecadienoate isomer could be further desaturated to 9(Z)-octadecen-12-ynoate (crepenynate) by the enzyme. The evolutionarily closely related Delta12 epoxygenase from Crepis palaestina had only weak desaturase activity but could also produce 9(Z),12(E)-octadecadienoate from oleate. The Delta6 acetylenase/desaturase from C. purpureus, on the other hand, produced only the 6(Z) isomers using C16 and C18 acyl groups possessing a Delta9 double bond as substrates. The Delta6 double bond was efficiently further converted to an acetylenic bond by a second round of desaturation but only if the acyl substrate had a Delta12 double bond and that this was in the Z configuration.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ⁹cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ⁶-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ⁶ desaturase to Δ⁹ regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ⁶ to Δ⁹ selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ⁹ to Δ⁶ selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ⁶ position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Predictions regarding the mechanism of Δ⁶ regioselectivity of the Thunbergia alata desaturase based on X-ray crystallography and molecular dynamics simulations are confirmed by experiment.     

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.     

Moth desaturase characterized that produces both Z and E isomers of delta 11-tetradecenoic acids

The redbanded leafroller moth, Argyrotaenia velutinana (Lepidoptera: Tortricidae) uses a 92:8 mixture of (Z)-11- and (E)-11-tetradecenyl acetate in its pheromone blend. These are produced in the abdominal pheromone gland from the corresponding acids, which are biosynthesized in the gland in a 3:2 Z/E ratio by desaturation of myristoyl CoA. The delta 11 desaturase involved in this reaction exhibits unusual substrate and stereospecificities in specifically producing Z11 and E11 isomers of tetradecenoic acid, and exhibiting no activity with C16 and C18 precursor acids. This report describes the cloning and expression of the redbanded leafroller moth delta 11 desaturase, and compares its amino-acid sequence to those of other known insect Z9, Z10, Z11, and E11 desaturases. The metabolic Z9 desaturase from fat body tissue also was cloned and expressed, and found mainly to produce Z9-16:Acid and Z9-18:Acid. The open reading frame of the delta 11 desaturase encodes a protein with 329 amino acids, whereas the open reading frame of the Z9 desaturase encodes a protein with 351 amino acids. Addition of this new delta 11 desaturase with its different substrate and regiospecificites to the databank of characterized integral-membrane desaturases will be key in efforts to determine amino-acid mutations responsible for the wide array of unsaturated fatty-acid products.     

A palmitoyl-CoA-specific delta9 fatty acid desaturase from Caenorhabditis elegans

Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the delta9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast delta9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast delta9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-5, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common delta9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.     

M?ssbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase.

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and M?ssbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The M?ssbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the M?ssbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the M?ssbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.     

Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.     

Fat metabolism in higher plants. The function of acyl thioesterases in the metabolism of acyl-coenzymes A and acyl-acyl carrier proteins.

Extracts of avocado mesocarp rapidly desaturate stearyl-acyl carrier protein (ACP) to free oleic acid. In addition to stearyl-ACP desaturase activity, the extracts contained a very active acyl thioesterase. After this activity was separated by ammonium sulfate fractionation from stearyl-ACP desaturase, over 95% of the desaturase product (18:1) was recovered as 18:1-ACP. The thioesterase was much more active toward 18:1-ACP than toward the other acyl-ACPs and acyl-CoAs tested. Long chain acyl thioesterase activity was present in a variety of plant cells, photosynthetic as well as nonphotosynthetic. The possible role of acyl thioesterases in regulating plant biosynthetic reactions involving lipids is discussed.     

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana , designated Ch FatB2 , has been identified, which, when expressed in Escherichia coli , provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1 , another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.