Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

Thompson, Gregory A.; Scherer, Donna E.; Aken, S Foxall-Van; Kenny, James W.; Young, Helen; Shintani, David K.; Kridl, Jean C.; Knauf, Vic

doi:10.1073/pnas.88.6.2578

Cited by 137 publications

(88 citation statements)

References 27 publications

(19 reference statements)

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“…(5,9). In contrast, it has been reported that the ⌬ 9 -18:0-ACP DES does not generate any detectable monounsaturated fatty acids when expressed in E. coli (28). These apparently contradictory findings are likely related to the relative pool sizes of acyl-ACPs.…”

Section: Characterization Of the Substrate Specificity Of Mutants Of contrasting

confidence: 42%

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Cahoon

Shanklin

2000

Proc. Natl. Acad. Sci. U.S.A.

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We demonstrate that naturally occurring C14 and C16-specific acylacyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA͞fadR mutant. Under the same growth conditions, C 18-specific ⌬ 9 -stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C 14 and C16 acyl-ACPs but a relative lack of C 18 acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor ⌬ 9 -18:0-ACP desaturase that display enhanced specificity for C 14 and C16 acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor ⌬ 9 -18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.A cyl-acyl carrier protein (ACP)-desaturases (DES) are a family of soluble enzymes that are associated with the synthesis of monounsaturated fatty acids in plants (1). These enzymes catalyze the insertion of a double bond into saturated fatty acids bound to ACP in the plastids of plant cells, and thus serve as one of the primary determinants of the unsaturated fatty acid content of plant membranes and seed storage oils. The most widely occurring member of this family is the ⌬ 9 -stearoyl (18:0 ‡ )-ACP DES, which introduces the double bond of oleic acid (1). In addition, a number of other acyl-ACP DES have been identified that are associated with the synthesis of unusual monounsaturated fatty acids and have altered substrate and regio-specifities relative to the ⌬ 9 -18:0-ACP DES. Variant acyl-ACP DES identified to date include the ⌬ 4 -palmitoyl (16:0)-ACP DES from Umbelliferae seed (2, 3), the ⌬ 6 -16:0-ACP DES from black-eyed Susan vine (Thunbergia alata) seed (4), the ⌬ 9 -myristoyl (14:0)-ACP DES from geranium (Pelargonium xhortorum) trichomes (5), and ⌬ 9 -16:0-ACP DES from cat's claw (Doxantha unguis-cati) (6) and milkweed (Asclepias syriaca) (7) seeds. These enzymes share Ն60% amino acid sequence identity with the ⌬ 9 -18:0-ACP DES.This collection of structurally related, but functionally diverged, enzymes provides a tool for understanding how acyl-ACP DES recognize the chain length of substrates and position the placement of double bonds. Characterization of these properties also has been greatly aided by information from the crystal structure of the castor ⌬ 9 -18:0-ACP DES (8). From the...

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Section: Characterization Of the Substrate Specificity Of Mutants Of contrasting

confidence: 42%

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Cahoon

Shanklin

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…Construction of a plasmid cDNA bank from mRNA of developing safflower embryos, insertion of the cDNA clones into XgtlO, and phage screening were as described (29 In vitro mutagenesis of safflower 18:1-ACP thioesterase clones 2-1 and 5-2 was performed using the method of Kunkel et al (10). The following oligonucleotides were designed to direct the insertion of SailI and NcoI sites immediately at the start of the mature protein coding region of cDNA clones 2-1 and 5-2, respectively: 3135, 5'-ACGGTGGCGCCGGTGTTGTCGACCATGGCGGTGA-GGACC-3'; 3134, 5'-GTTTCTCCGTTGCTAATGTCGA-CCATGGCGGTGGCGACCGGA-3'.…”

Section: Library Construction and Screeningmentioning

confidence: 99%

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Knutzon¹,

Bleibaum²,

Nelsen³

et al. 1992

Plant Physiol.

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Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34-and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50-to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16-and 18-carbon acyl-ACP substrates.Fatty acids are important components of membrane phospholipids in all plant tissues and are also stored in the form of triacylglycerols in the seeds of many species for use as an energy source upon germination. Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) nia bay (Umbellularia californica), which accumulates high levels of lauric acid (12:0) in its seed triacylglycerols. Recently, a 12:0-ACP-specific thioesterase has been isolated from this species and shown to redirect fatty acid synthesis to the production of laurate in transgenic plants (6,18,30). In plants that accumulate the longer-chain fatty acids, the specificity of the acyl-ACP thioesterase may also play a role in determining the final fatty acid composition of the storage oil.We are interested in studying acyl-ACP thioesterases with specificities for 16-and 18-carbon fatty acids. Acyl-ACP thioesterase activities have been characterized at various stages of purification from a number of plant species that accumulate 16-and 18-carbon fatty acids in their triacylglycerols, including avocado (17, 24), safflower (Carthamus tinctorius) (12), squash (8), and rapeseed (7). The enzymes show distinct preferences for 18:1-ACP over 18:0-ACP and 16:0-ACP substrates and are relatively inactive on acyl-CoAs.To investigate further the role of acyl-ACP thioesterase in the determination of seed oil composition, we have purified and obtained partial amino acid sequence of an 18:1-ACP thioesterase from developing safflower seeds. Two different classes of cDNA clones encoding 18:1-ACP thioesterase have been isolated and characterized. The substrate specificity of the protein encoded by each clone has been examined by ...

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“…Fatty acid acyl carrier protein (ACP) 3 desaturases (EC 1.14.99.6) convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction (1)(2)(3)(4)(5)(6). ⌬ 9 -desaturation of 18:0-ACP 4 occurs ubiquitously in the plastids of plants and thus plays an important role in determining the fluidity of cell membranes (7).…”

mentioning

confidence: 99%

The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

Guy

Whittle

Kumaran

et al. 2007

Journal of Biological Chemistry

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The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the ⌬ 4 desaturation of 16:0-ACP and the ⌬ 9 desaturation of 18:0-ACP and further desaturates ⌬ 9 -16:1 or ⌬ 9 -18:1 to the corresponding ⌬ 4,9 dienes. The crystal structure of the enzyme has been solved to 1.95 Å resolution, and both the iron-iron distance of ϳ3.2 Å and the presence of a -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. Fatty acid acyl carrier protein (ACP)3 desaturases (EC 1.14.99.6) convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction (1-6). ⌬ 9 -desaturation of 18:0-ACP 4 occurs ubiquitously in the plastids of plants and thus plays an important role in determining the fluidity of cell membranes (7). Plants can express distinct acyl ACP desaturases, e.g. Arabidopsis thaliana has a family of seven acyl-ACP desaturases that are differentially expressed in different tissues (8). A structurally related homolog with unknown function from Mycobacterium has also been described (9).Seed oils of various plants accumulate monounsaturated fatty acids with unusual chain lengths and/or double bond positions. Several ⌬ 9 desaturases that recognize 14-, 16-, and 18-carbon chain lengths (4, 10 -12) have been described, and others with ⌬ 6 and ⌬ 9 regiospecificities have also been reported (13,14). The biosynthetic origin of unusual fatty acids is not always straightforward; for instance, Coriandrum sativum L. (coriander) and Hedera helix (English ivy) accumulate petroselinic acid (18:1⌬ 6 ) that originates via a ⌬ 4 desaturation of 16:0-ACP followed by 2-carbon elongation to 18:1⌬ 6 (15). The genes encoding these ⌬ 4 -16:0-ACP desaturase enzymes have been isolated (15,16), and their amino acid sequences are homologous to that of the castor ⌬ 9 -18:0-ACP desaturase for which the crystal structure has been determined (17,18). This implies that ac...

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Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

Cited by 137 publications

References 27 publications

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

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