Proc. Natl. Acad. Sci. USAVol. 88, pp. 2578-2582, March 1991Biochemistry

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

(fatty acid biosynthesis/acyl carrier protein/transit peptide/unsaturated fatty acids)

GREGORY A. THOMPSON*, DONNA E. SCHERER, SUSAN FOXALL-VAN AKEN, JAMES W. KENNYt,HEATHER L. YOUNGt, DAVID K. SHINTANI§, JEAN C. KRIDL, AND VIC C. KNAUFCalgene, Inc., 1920 Fifth Street, Davis, CA 95616

Communicated by Paul K. Stumpf, December 26, 1990 (received for review November 5, 1990)

ABSTRACT Stearoyl-acyl carrier protein (ACP) desatu-rase (EC 1.14.99.6) catalyzes the principal conversion of sat-urated fatty acids to unsaturated fatty acids in the synthesis ofvegetable oils. Stearoyl-ACP desaturase was purified fromdeveloping embryos of safflower seed, and extensive amino acidsequence was determined. The amino acid sequence was usedin conjunction with polymerase chain reactions to clone afull-length cDNA. The primary structure of the protein, asdeduced from the nucleotide sequence of the eDNA, includes a33-amino-acid transit peptide not found in the purified enzyme.Expression in Escherichia coli of a gene encoding the matureform of stearoyl-ACP desaturase did not result in an alteredfatty acid composition. However, active enzyme was detectedwhen assayed in vitro with added spinach ferredoxin. The lackof significant activity in vitro without added ferredoxin and thelack of observed change in fatty acid composition indicate thatferredoxin is a required cofactor for the enzyme and thatE. coliferredoxin functions poorly, if at all, as an electron donor forthe plant enzyme.

Membrane fluidity and function are greatly influenced by theratios of saturated to unsaturated fatty acids in the membranelipids. In plants (1) and bacteria (2), the saturated fatty acidsare synthesized in two-carbon increments as acyl thioestersof acyl carrier protein (ACP). In enteric bacteria such asEscherichia coli, the primary unsaturated fatty acids arecis-All C18:1 (vaccenic acid) and cis-A9 C16:1 (palmitoleicacid). Vaccenic and palmitoleic acids are synthesized byelongation of precursor monounsaturated acyl-ACPs; thesaturated 16- and 18-carbon fatty acids (palmitic and stearicacids) are synthesized from precursor saturated acyl-ACPs.In higher plants, however, the unsaturated 16-carbon trans-hexadec-9-enoic acid and 18-carbon oleic acid (cis-A9 C18:1)are formed directly from palmitic and stearic acids esterifiedto specific glycerol lipids or to ACP (3). These reactions takeplace in the chloroplast (or proplastid in nonphotosynthetictissues). Further double bonds are introduced into the mono-unsaturated acyl-lipids, typically at the 12 position followedby desaturation at the 15 or 6 positions of the diunsaturatedspecies; saturated acyl groups generally do not serve assubstrates for desaturation at the 6, 12, or 15 position in thecarbon chain. Thus in higher plants, the ratio of saturatedfatty acids to unsaturated fatty acids in membrane lipids isdirectly regulated by the enzymes that catalyze the conver-sion of saturated species to monounsaturated ones.Our interests lie in the regulation of the enzymatic steps in

higher plants that determine the relative amounts of specificsaturated and unsaturated fatty acids in neutral storage lipids.Unsaturated fatty acids in seed oils are predominantly 18

carbons or more in length and are derived by a series ofenzymatic steps following the conversion of stearoyl-ACP tooleoyl-ACP. Stearoyl-ACP desaturase (EC 1.14.99.6) istherefore a necessary enzyme in the synthesis of unsaturatedfatty acids in seed oils. In this study, we describe thepurification of stearoyl-ACP desaturase from immature saf-flower embryos, determination ofamino acid sequence oftheenzyme, isolation of a corresponding cDNA, and derivationof the primary structure of both the precursor and matureenzymes.¶ Through use of the cloned gene to express anactive enzyme in E. coli, we confirm that the gene encodesstearoyl-ACP desaturase and show that ferredoxin is a nec-essary cofactor (4, 5).

MATERIALS AND METHODSMaterials. Chemicals and cofactors used in the enzyme

assay, including bovine serum albumin, catalase (twice crys-talized from bovine liver), spinach ferredoxin, ferredoxin:NADP+ oxidoreductase (spinach leaf), and NADPH, werefrom Sigma. [9,10(n)-3H]Stearic acid was obtained by reduc-tion of [9,10(n)-3H]oleic acid (NEN) with hydrazine hydrateas described by Johnson and Gurr (6). ACP was isolated fromE. coli strain K-12 (Grain Processing, Muscatine, Iowa) asdescribed by Rock and Cronan (7). The desaturase assaysubstrate, [9,10(n)-3H]stearoyl-ACP (100 mCi/mmol; 1 Ci =37 GBq), was prepared from [9,10(n)-3H]stearic acid and E.coliACP by using the enzymatic synthesis procedure ofRocket al. (8).Enzyme Assay. Stearoyl-ACP desaturase activity was de-

termined by measuring the enzyme-catalyzed release oftritium from [9,10(n)-3H]stearoyl-ACP as described by Ta-lamo and Bloch (9). Each assay was prepared by mixing 5 ILIof dithiothreitol (100 mM), 10 .ul of bovine serum albumin (10mg/ml), 15 ILI ofNADPH {25 mM, freshly prepared in 0.1 MN-[tris(hydroxymethyl)methyl]glycine (Tricine) HCl, pH8.2}, 25 .l of spinach ferredoxin (2 mg/ml), 3 1.l of ferre-doxin:NADP+ oxidoreductase (2.5 units/ml), 1 .l1 of catalase(800,000 units/ml), and 150 p.l of water. After 10 min at roomtemperature, this mixture was added to a 13 x 100 mmscrew-cap test tube containing 250 p.l of sodium 1,4-

Abbreviations: ACP, acyl carrier protein; IPTG, isopropyl f-D-thiogalactopyranoside.*To whom reprint requests should be addressed.tPresent address: Beckman Center, Stanford University MedicalCenter, Stanford, CA 94305-5425.tPresent address: Department of Human Physiology, School ofMedicine, University of California, Davis, CA 95616.§Present address: Department of Botany and Plant Pathology, PlantBiology Laboratory, Michigan State University, East Lansing, MI48824.$The sequence reported in this paper has been deposited in theGenBank data base (accession no. M61109).

2578

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Proc. Natl. Acad. Sci. USA 88 (1991) 2579

piperazinediethanesulfonate (Pipes; 0.1 M, pH 6.0), and 10 Alofthe enzyme sample to be assayed. The reaction was startedby addition of substrate, 30 pl of [9,10(n)-3H]stearoyl-ACP(10 ,uM in 0.1 M sodium Pipes, pH 5.8), and after the tube hadbeen sealed with a cap, allowed to proceed for 10 min withshaking at 230C. The reaction was terminated by addition of1.2 ml of 5.8% trichloroacetic acid, and the resulting precip-itated acyl-ACPs were removed by centrifugation. A 500-pIaliquot of the aqueous fraction was neutralized with 1 M Trisbase, and the tritium released by the desaturase reaction wasmeasured by liquid scintillation spectrometry. A unit ofactivity is defined as the release of 4 ,umol of 3H per min,equivalent to the conversion of 1 ,umol of stearoyl-ACP tooleoyl-ACP.

Purification of Stearoyl-ACP Desaturase. Immature seedswere collected from greenhouse-grown safflower plants be-tween 16 and 18 days after the appearance of the first flowerpetals; the collected seed were frozen in liquid nitrogen andstored at -70'C. Extraction and purification of the desatu-rase from frozen tissue were performed essentially as de-scribed by McKeon and Stumpf (5). This entailed grindingand production of an acetone powder, followed by triturationof the powder in phosphate buffer and chromatographythrough DEAE-cellulose (Whatman DE52), and ACP-Sepharose 4B. To remove traces of a seed albumin whichcarried through the first purification steps, a reverse-phaseHPLC step was added to the protocol. The pooled fractionscontaining desaturase activity from ACP-Sepharose wereapplied directly to a Vydac C4 reverse-phase column (0.45 x15 cm) equilibrated in 0.1% trifluoroacetic acid/7% (vol/vol)acetonitrile, and after a 10-min wash with equilibration bufferthey were eluted with a gradient of increasing acetonitrile(7-70%, vol/vol) in 0.1% trifluoroacetic acid over a period of45 min; the flow rate throughout was 0.5 ml/min. Elutingcomponents were monitored by absorbance at 214 nm. Thecontaminating seed albumin was eluted at 28 min (approxi-mately 30% acetonitrile), and the desaturase protein waseluted at 42 min (approximately 50% acetonitrile). The en-zyme was not active following this step, and it was identifiedas a single 40-kDa band on SDS/PAGE performed as de-scribed by Laemmli (10). Approximately 50 ,ug of purestearoyl-ACP desaturase protein was obtained from 50 g offrozen seed tissue.Primary Structure Determination. Purified stearoyl-ACP

desaturase was reduced with dithiothreitol and alkylated withiodo[3H]acetic acid (64 Ci/mol, NEN) to ensure more com-plete denaturation prior to digestion and to aid in identifica-tion of cysteine residues. Alkylated protein samples wereproteolytically digested with sequencing grade trypsin, en-doproteinase Lys-C, endoproteinase Glu-C, or endoprotein-ase Asp-N under conditions recommended by the supplier(Boehringer Mannheim). Peptides obtained from each of thedigests were separated by reverse-phase HPLC. The first-dimension peptide map was obtained from a Vydac C18reverse-phase column (0.2 x 15 cm) equilibrated in 7%acetonitrile in 0.1 mM sodium phosphate, pH 2.2; afterwashing for 20 min with the equilibration buffer, peptideswere eluted with a gradient of increasing acetonitrile (7-70%)in 0.1 M sodium phosphate, pH 2.2, over 120 min at a flowrate of 50 pl/min. Eluted components were monitored byabsorbance at 214 nm, and individual peptide peaks werecollected as separate fractions. When there was sufficientmaterial in an individual peak and contamination with otherpeptides was suspected, further purification was achieved byapplication to a second Vydac C18 reverse-phase column (0.2x 15 cm) equilibrated in 7% acetonitrile in 0.1% trifluoro-acetic acid. Again, after a 20-mmn wash with equilibrationbuffer, pure peptides were eluted with a gradient (7-70%) ofacetonitrile in 0.1% trifluoroacetic acid over 120 min at a flowrate of 50 pl/min. The purified peptides were subjected to

amino acid sequence analysis by automated Edman degra-dation on an Applied Biosystems model 477A pulsed-liquidsequencer with on-line phenylthiohydantoin (PTH) aminoacid analysis. N-terminal amino acid sequence informationwas obtained by analysis of an undigested sample on thesequencer.Library Construction and Screening. mRNA was isolated

from polyribosomes of developing safflower seeds by themethod of Jackson and Larkins (11) as modified by Goldberget al. (12). A cDNA library was made in the plasmid cloningvector pCGN1703 as described (13). pCGN1703 is similar tovectors described by Alexander (13) but contains differentpolylinker cloning sites (14). The cDNA bank containedapproximately 4 x 106 clones with an average cDNA insertsize of 1000 base pairs. The bank was amplified by growinga culture of pooled E. coli containing the cDNA clones andisolating plasmid DNA. This DNA served as template forpolymerase chain reactions (PCRs) with degenerate primers(15). Oligonucleotide primers were synthesized on an Ap-plied Biosystems DNA synthesizer model 380A. Reactionswere run on a Perkin-Elmer/Cetus DNA thermal cyclerusing a thermocycle program of 1 min at 940C, 2 min at 420C,and a 2-min rise from 420C to 720C for 30 cycles; followed by10 cycles of 1 min at 940C, 2 min at 420C, and 3 min at 720Cwith increasing 15-sec extensions of the 72°C step; and a final10-min 72°C extension.To facilitate library screening, plasmid DNA from the

safflower cDNA bank was digested with the enzyme EcoRIand ligated into EcoRI-predigested and phosphorylated AgtlOarms (Stratagene). Cloning ofDNA sequences, radiolabelingof DNA, and screening of phage libraries were done usingstandard techniques (16). DNA sequence was determined bythe method of Sanger et al. (17) using dideoxynucleotides;both strands were sequenced. DNA sequence data wereanalyzed by using IntelliGenetics software.E. coli Expression Analysis. Stearoyl-ACP desaturase was

subcloned in the E. coli expression vector pET8c (18). Themature coding region (plus an extra Met codon) of thedesaturase cDNA with accompanying 3' sequences wasinserted as an Nco I-Sma I fragment into pET8c at the NcoI and BamHI sites (the BamHI site had been treated with theKlenow fragment ofDNA polymerase) to create pCGN3208.The plasmid pCGN3208 was maintained in E. coli strainBL21 (DE3), which contains the T7 polymerase gene underthe control of the isopropyl f3-D-thiogalactopyranoside(IPTG)-inducible lacUV5 promoter (18).

E. coli cells containing pCGN3208 were grown at 37°C toan OD595 of -0.7 in NZYM broth (16) containing 0.4%glucose and 300 mg of penicillin per liter, and then induced for3 hr with 0.4 mM IPTG. Cells were pelleted from 1 ml ofculture, dissolved in 125 ,ul of SDS sample buffer (10), andheated to 100°C for 10 min. Samples were analyzed bySDS/PAGE at 25 mA for 5 hr. Gels were stained in 0.05%Coomassie brilliant blue/25% (vol/vol) isopropyl alcohol/10% (vol/vol) acetic acid.For activity assays, cells were pelleted at 14,800 x g for 15

min, resuspended in 20 mM potassium phosphate at pH 6.8,and broken in a French press apparatus at 16,000 psi (110MPa). Debris was pelleted at 5000 X g for 5 min. Thesupernatant fraction was applied to a Sephadex G-25 cen-trifugal gel filtration column (Boehringer Mannheim) equili-brated with 20 mM potassium phosphate at pH 6.8. Columnswere centrifuged for 4 min at 5000 x g. The effluent wascollected and used as enzyme source in the desaturase assaydescribed above. k

RESULTS AND DISCUSSIONPurification and Sequencing of Stearoyl-ACP Desaturase. A

previously published protocol (5) for the purification of

Biochemistry: Thompson et al.

2580 Biochemistry: Thompson et al.

Fl: ASTLGSSTPKVDNAKKPFQPPREVHVQVTHSMPPQKIEIFKSIEGWAEQNILVHLKPVEKCF2:--------D E QK EDDT TTR.

F2: DFLPDPASEGFDEQVKELRARAKE IPDDYFVVLVGDMITEEALPTYQTMLNTL

F3: D--GASLTPWAVWT

F5: DM-QIQKTIQYLI

F4: DLL-TYLYL-GRV- II

F6: TENSPYLGFIYTSFQER

F7: DVKLAQICGTIASD-KR-ETAYTKIVEKLFEIDPDGTVLAFADMMRKKISMPAHLMY

--LQ QQEQQ

F8: DNLF F9: D-FSAVAQRLGVYTAK_T_

I

F10: DYADILEFLVGR-K

F11: VADLTGLSGEGRKAQDYVCGLPPRIRRLEERAQGRAKEGPVVPFSWIFDRQ\

= Lys-C = Glu-C = Asp-N = Tryl

stearoyl-ACP desaturase from safflower seeds involved theuse of acetone powder extraction, anion-exchange chroma-tography, and affinity chromatography on ACP-Sepharose.This protocol resulted in a 900-fold increase in specificactivity over the acetone powder extract, and the productappeared homogeneous by standard SDS/PAGE analysis(10). However, analysis by gel electrophoresis under nonre-ducing conditions or by reverse-phase HPLC revealed thepresence of a major contaminant. Peptide sequence homol-ogy to other albumins (unpublished results) indicated that thecontaminant was a seed-storage albumin. Reverse-phaseHPLC was used to remove the contaminant so that the finalpreparation contained a single silver-stained protein band ofapproximately 40 kDa by SDS/PAGE, with or without re-ducing agent. It was not possible to identify this proteinpositively as desaturase or to determine specific activitybecause activity was lost during this step.

Partial amino acid sequence of the purified 40-kDa proteinwas determined from four separate proteolytic digests. Fig.1 shows these sequences assembled into 11 fragments byusing overlapping peptides from the different digests. Frag-ment F1 was deduced to be the N-terminal fragment as twodifferent proteolytic cleavages, with Lys-C and Glu-C, eachyielded peptides beginning with the same sequence (Ala-Ser-Thr-Leu-Gly-Ser-Ser-Thr-Pro-Lys). This deduction was sup-ported by N-terminal sequence data from undigested en-zyme, although extremely low initial yields in the Edman

A

FIG. 1. Amino acid sequence determined for pu-rified safflower stearoyl-ACP desaturase. Each frag-ment (F1 to F11 in order found in the cDNA) repre-sents a synthesis of sequence information from pep-tides originating from different proteolytic digests(endoproteinase Lys-C, endoproteinase Glu-C, en-doproteinase Asp-N, trypsin), which have beenmatched and aligned as indicated, and then orderedas they ultimately were found in the cDNA (see Fig.3). The bars representing each peptide have differentshading patterns as indicated, to show their proteo-lytic origins. Some bars are interrupted to show aspecific amino acid; this is to indicate positions wherea second amino acid was detected, or where therewere differences between peptides that otherwise hadthe same sequence. Such multiplicities or differencesmight arise from impurities in the peptides or frommicroheterogeneity of the protein. The IUPAC one-letter code for amino acid residues has been used. Ahyphen indicates a cycle in which it was not possibleto identify a residue, either because no signal wasdetected or because there were too many signals inthat position.

degradation suggested that most of the protein was blockedat the N terminus.

Cloning of a cDNA for Safflower Embryo Stearoyl-ACPDesaturase. mRNA was isolated from safflower embryoscollected 14-17 days after flowering. During this period ofembryo development, seed oil was accumulating in thecotyledons and the stearoyl-ACP desaturase activity waseasily detected in crude extracts. A cDNA bank was pre-pared from this mRNA by the method of Alexander (13).Amplified DNA from this bank was utilized as templates forPCR experiments.PCRs were used to generate a DNA encoding part of

stearoyl-ACP desaturase. Oligonucleotide primers were de-signed from amino acid sequences contained in the peptidefragment F2 (Fig. 2). The PCR product including the primerswas predicted to be 107 base pairs, sufficiently large to detecton a 1.5% agarose gel. Separate reactions utilizing combina-tions of the degenerate sense and antisense primers resultedin products of the expected size. This indicated that exacthomology may not have been necessary for initiation of thePCR. DNA sequence analysis of a cloned product from the13-4/13-5a PCR reaction indicated that the amplified DNAencoded the F2 peptide region as shown in Fig. 2. A 50-nucleotide sequence (DESAT-50: 5'-GTAAGTAGGT-AGGGCTTCCTCTGTAATCATATCTCCAACCAAAA-CAACAA-3') matching the noncoding strand of part of thePCR product was synthesized to screen the safflower embryocDNA bank.

K E I P D D Y FVVLVGDM.ITE-ALPTY Q T M L N T

B AAAGAAAUUCCUGAUGAUUAU

G G C C CC C

A A

CAAACUWG C

A

G

DESAT 13-1: 5'- GCTAAGCTTAAPGAPATBCCAGAQGAQTA -3'

c DESAT 13-2: 5'- GCTAAGCTTAAPGAPATBCCGGAQGAQTA -3'

DESAT 13-3: 5'- GCTAAGCTTAAPGAPATBCCCGAOCAQTA -3'

DESAT 13-4: 5'- GCTAAGCTTAAPGAPATBCCTGAQGAQTA -3'

D 3'- GTQTGNTACGANTTPTGCTTAAGCGA -5'

33'- GTQTGNTACAAQTTPTGCTTAAGCGA -5'

NUGCUUAAUACUC C C FIG. 2. Design of oligonucleotide primers forA A PCRs. (A) Amino acid sequence (one-letter code)G C from fragment F2 (see Fig. 1). (B) Codon degener-

UUA acy corresponding to regions of peptide sequenceG used to design the PCR primers. (C) Sequence of

four oligonucleotide pools (13-1 to 13-4) used toprime in the sense direction. Nine extra bases wereadded to the 5' terminus to include a HindIII site.

- ~ (D) Sequence of two oligonucleotide pools (13-Saand 13-6a) used to prime in the antisense direction.Nine extra bases were added to the 5' terminus toinclude an EcoRI site. Designations for nucleotide

DESAT 13-Sa degeneracy: P = A and G; Q = T and C; N = A, C,DESAT 13-6a T, and G; B = C, T, and A.

Proc. Natl. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 2581

129GCTCACTTGTGTGGTGGAGGAGAAAAACAGACTCACAAAOACTTTGCGACTGCCAAGAACAACAACAACAACAAGATCAAGMGAAGAAGAAGAAGATCAAAA ATG GCT CTT CGA ATC ACT CCA GTG

MET Ala Leu Arg Ile Thr Pro ValXool 237

ACC TTG CAA TCG GAG AGA TAT CGT TCG TTT TCG TTT CCT AAG AAG GCT AAT CTC AGA TCT CCC AAA TTC GCC ATM GCC TCC ACC CTC GGA TCA TCC ACA CCG AAG GTTThr Leu Gln Ser Glu Arg Tyr Arg Ser Ph. Ser Phe Pro Lys Lys Ala Asn Leu Arg Ser Pro Lys Phe Ala MET AIA Ser Thr Leu Gly Ser Ser Thr Pro Lys Val

345GAC AAT GCC AAG AAG CCT TTT CAA CCT CCA CGA GRG GTT CAT GTT CAG GTG ACG CAC TCC ATG CCA CCA CAG AMG ATA GAG ATT TTC AAA TCC ATC GAG GGT TGG OCTAsp Asn Ala Lys Lys Pro Phe Gin Pro Pro Arg Glu Val His Val Gln Val Thr His Ser MET Pro Pro Gln Lys Ile Glu Ile Phe Lys Ser Ile Glu Gly Trp Ala

_______ __ __ ___ - --Arg ---

453GAG CAG AAC ATA TrG GTT CAC CTA AAG CCA GTG GAG AAA TGT TGG CMA GCA CAG GAT TTC TTG CCG GAC CCT GCA TCT GAA GGA TTT GAT GAA CAA GTC AM GM CTAGlu Gln Asn Ile Loeu Val His Leu Lys Pro Val Glu Lys Cys Trp Gln Ala Gin Asp Phe Leu Pro Asp Pro Ala Ser Giu Gly Phe Asp Glu Gln Val Lys Glu Leu.--------------- - -- -- -- -- Thr - - -

561AGG GCA AGA GCA AAG GAG ATT CCT GAT GAT TAC mTr GTT GTT TTG GTT GGA GAT ATG ATT ACA GAG GAPA 0CC CTA CCT ACT TAC CAA ACA ATG CTT AAT ACC CTA GATArg Ala Arg Ala Lys Glu Ile Pro Asp Asp Tyr Phe Val Val Leu Val Gly Asp MET Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr MET Leu Asn Thr Leu Asp

669GGT GTA COT GAT GAG ACT GGG GCT AGC CTT ACG CCT TGG OCT GTC TOG ACT AGG GCT TOG ACA GCT GAA GAG AAC AGG CAT GGC GAT CTT CTC CAC ACC TAT CTC TACGly Vai Arg Asp Glu Thr Gly Ala Ser Leu Thr Pro Trp Ala Val Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg His Gly Asp Leu Leu His Thr Tyr Lou Tyr

777CTT TCT GGG COGG GTA GAC ATG AGG CAG ATA CAG AAG ACA ATT CAG TAT CTC ATT GGG TCA GGA ATG GAT CCT CGT ACC GMA AAC AGC CCC TAC OTT GGG TTC ATC TACLeu Ser Gly Arg Val Asp MET Arg Gin Ile Gln Lys Thr Ile Gln Tyr Leu Ile Gly Ser Gly MET Asp Pro Arg Thr Glu Asn Ser Pro Tyr Lou Gly Phe Ile Tyr-- ----le----- -------- --- ----

885ACA TCG TTm CAA GAG CGT GCC ACA TTT GTT TCT CAC GGA AAC ACC GCC AGG CAT GCA AAG GAT CAT GGG GAC GTG AAA CTG GCG CAA ATT TGT GGT ACA ATC GCG TCTThr Ser Phe Gln Glu Arg Ala Thr Phe Val Ser His Gly Asn Thr Ala Arg His Ala Lys Asp His Gly Asp Val Lys Leu Ala Gln Ile Cys Gly Thr Ile Ala Ser

993GAC GAA AAG CGT CAC GAG ACC GCT TAT ACA AAG ATA GTC GAA AAG CTA TTC GAG ATC GAT CCT GAT GGC ACC GTT CTT GCT TTT GCC GAC ATG ATG AGG AAA AAG ATCAsp Glu Lys Arg His Glu Thr Ala Tyr Thr Lys Ile Val Glu Lys Leu Phe Glu Ile Asp Pro Asp Gly Thr Val Leu Ala Phe Ala Asp MET MET Arg Lys Lys Ile-- Leu Gln --------------------Glu -- Gln ----- -----

1101TCG ATG CCC GCA CAC TTG ATG TAC GAT GGG CGT GAT GAC AAC CTC TTC GAA CAT TTC TCG GCG GTT GCC CAA AGA CTC GGC GTC TAC ACC GCC AAA GAC TAC GCC GACSer MET Pro Ala His Leu MET Tyr Asp Gly Arg Asp Asp Asn Leu Phe Glu His Phe Ser Ala Val Ala Gln Arg Leu Gly Val Tyr Thr Ala Lys Asp Tyr Ala Asp---------- --- -- ----- --AAsp -- Leu ------ -----

1209ATA CTG GAA TTT CTG GTC GGG CGG TGG AAA GTG GCG GAT TTG ACC GOC CTA TCT GGT GAA G0G CGT AAA GCG CAA GAT TAT GTT TGC GGG TTG CCA CCA AGA ATC AGAIle Leu Glu Phe Leu Val Gly Arg Trp Lys Val Ala Asp Leu Thr Gly Leu Ser Gly Glu Gly Arg Lys Ala Gln Asp Tyr Val Cys Gly Leu Pro Pro Arg Ile Arg

1323AGG CTG GAG GAG AGA GCT CAA G0G CGA GCA AAG GAA GGA CCT GTT GTT CCA TTC AGOC TGG ATT TTC GAT AGA CAG GTG MG CTG TGArg Leu Glu Glu Arg Ala Gln Gly Arg Ala Lys Glu Gly Pro Val Val Pro Phe Ser Trp Ile Phe Asp Arg Gln Val Lys Lou

3A AGAAAAAAAAAACGAGCAGTGAGTICG

1466

TATrTATAGAACTCGTTrATGCCAATTGATGAcGTA cGTC GOTO1-1r-r1TrATTGT 1533

FIG. 3. DNA sequence of the 1533-base cDNA of a stearoyl-ACP desaturase mRNA from developing safflower embryos. The Nco Irestriction site used to adapt the gene for expression of desaturase enzyme in E. coli is indicated. The underlined Ala residue indicates the Nterminus ofthe mature form of the enzyme as suggested by peptide sequence. Matching peptide sequence is indicated by hyphens (--- -); positionsof ambiguity or mismatch with respect to peptide sequence are indicated by the other amino acid found in the peptides (see Fig. 1). Suchsequences may indicate the possibility of other desaturase genes, or they may be from minor contaminants in the sequenced peptides.

Forty-five thousand phage plaques were screened with theoligonucleotide probe DESAT-50. Of 16 plaques hybridizingto the probe, 2 were isolated and characterized as cDNAclones in pCGN1703. Both clones represented mRNAs tran-scribed from the same gene on the basis of identical 3'untranslated DNA sequences. The longer of these, pCGN-2754, was completely sequenced from both strands. The1533-base-pair DNA sequence, not including the 3' poly(A)sequence, is shown in Fig. 3. A single open reading frameaccounts for all of the desaturase peptide sequences. The Metcodon at nucleotide 106 of the cDNA resembles a consensustranslational start (19) and lies 99 bases 5' to the sequenceencoding the N terminus of the mature enzyme. Sincestearoyl-ACP is in the proplastid organelle (1), we anticipatedthat the cDNA would encode a precursor protein with atransit peptide (20) to translocate the desaturase enzyme intoproplastids. Thus, the 99 bases at the 5' end of the openreading frame encode the expected transit peptide with aprocessing site after the Met at residue 33. The cDNAsequence indicates that the mature enzyme is a 363-amino-acid peptide with a calculated molecular mass of 41,202 Da.

Verification that pCGN2754 Encodes Stearoyl-ACP Desat-urase. A unique Nco I restriction site in pCGN2754 includesthe Met codon preceding the transit processing site. Thephasing of the Nco I site allowed subcloning of DNA se-quences encoding the mature stearoyl-ACP desaturase in theunique Nco I sites ofE. coli expression vector pET8c to makepCGN3208. The pET8c system utilizes the phage T7 poly-merase enzyme to express genes inserted between a T7

promoter and transcription terminator (18). The predicted40-kDa protein was easily discerned in gel profiles of totalproteins from E. coli containing pCGN3208 (Fig. 4).

E. coli synthesize small amounts of stearoyl lipids. Giventhe expression levels of the desaturase protein, we antici-pated that strains containing the pCGN3208 plasmid mightcontain oleoyl lipids. No alteration of fatty acid composition

pET8c 3208

U I I U

97.4-66.2-

42.7 -31.0-'U.- 1-iOw.

* W21.0-ar

14.4

FIG. 4. SDS/PAGE analysisof safflower stearoyl-ACP desatu-rase expressed in E. coli. Extractsof uninduced (U) and induced (I)E. coli strain BL21(DE3) contain-ing either pET8c or pCGN3208were prepared as described in thetext. Molecular masses of stan-dards are indicated in kDa on theleft side. Stearoyl-ACP desaturaseis indicated by the arrow.

Biochemistry: Thompson et al.

2582 Biochemistry: Thompson et al.

Table 1. Expression of stearoyl-ACP desaturase activity inextracts from E. coli transformed with pET8c or pCGN3208

Activity, pmol/min per mg of protein

Uninduced IPTG-induced

Without With Without WithPlasmid Fd Fd Fd Fd

pET8c 1 0 0 5pCGN3208 7 1409 61 8609

Fd, reduced spinach ferredoxin.

in E. coli containing pCGN3208 as compared to E. colicontaining pET8c was observed (data not shown). However,sufficient levels of stearoyl-ACP as substrate may not havebeen present or accessible to the enzyme, or perhaps oleoyl-ACP is unstable in E. coli. When extracts of E. coli strainswere assayed for stearoyl-ACP desaturase activity, negligibleactivity was detected. However, when spinach ferredoxinwas added to the in vitro assay, significant amounts ofdesaturase activity were clearly demonstrated in extractsfrom E. coli containing pCGN3208 (Table 1), even in theabsence of IPTG for full induction of the T7 polymerase.These experiments verify that the cloned insert of

pCGN2754 does encode the stearoyl-ACP desaturase andthat the protein purified from safflower embryos by theprotocol of McKeon and Stumpf (5) represents authenticstearoyl-ACP desaturase.Requirement for Ferredoxin and Similarity to Other Desat-

urases. Minimal activity was detected in E. coli extractsunless spinach ferredoxin was added exogenously. More-over, even though (i) our enzyme assay uses stearoyl-ACP inwhich the ACP is isolated from E. coli, (ii) E. coli synthesizesstearoyl-ACP at least in trace amounts (2), and (iii) oleate canbe incorporated into E. coli lipids (21), we can detect no oleicacid in E. coli expressing relatively high levels of stearoyl-ACP desaturase. E. coli cells do contain ferredoxin, but itsfunction and structure are not well understood (22). Appar-ently, E. coli ferredoxin is not an efficient electron donor forthe safflower stearoyl-ACP desaturase. This apparent spec-ificity for plant ferredoxins could be tested by adding purifiedferredoxins from E. coli and other sources to extracts from E.coli containing pCGN3208.We detected no significant homology between the saf-

flower stearoyl-ACP desaturase amino acid sequence andthat of the rat stearoyl-CoA desaturase (23), which catalyzesthe analogous conversion of stearoyl-CoA to oleoyl-CoA.This suggests independent evolutionary origins for theseenzymes. Other "oxygen-requiring" desaturases are alsofound in cyanobacteria (24) and certain strains of Pseudo-monas (25); it will be of interest to see if these resemble thestearoyl-ACP desaturase of higher plants and if forms offerredoxin found in these prokaryotes can serve as functionalelectron donors for stearoyl-ACP desaturase. The otherdesaturases in plants appear to be membrane-bound (1); theirsimilarity to the soluble stearoyl-ACP desaturase has notbeen shown.

Plasmid pCGN3208 provides the means to produce notonly native enzyme but also enzymes modified by site-specific mutagenesis ofthe cloned gene. These studies will beessential to understand active site mechanisms and the rela-

tionship of structure to function for desaturase enzymes. Inaddition, cloned genes will allow alterations of in vivo levelsof enzyme activity in transgenic plants (26-29) for examina-tion of the role of stearoyl-ACP desaturase in determiningtotal unsaturated fatty acid content.

We are indebted to Burt Rose and Neal Ridge for DNA sequenceanalysis, to David M. Stalker, Don Lucas, Bill Hiatt, and MaelorDavies for critically reading the manuscript before submission, andto Paul Stumpffor many fruitful discussions of this work while it wasin progress.

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