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Biochimica et Bio$~ysicz Acia 1212 (19949 134-135

(Received IO December 1993; revised manuscript accepted 8 February 1994)

A cDNA for the oleoyl-aeyl carrier protein @L$@$)hioesterase (EC. X1.2.14) from coriander seed e~aos~erm ~~~~~~~~~~~~~~~CXUTI)as isolated using a safflower oleoyl-AGP thioesterase cDNA probe. The cotiander c NA coded for a 42.3 kiDa proteinin&ding a putative 40 amino acid plastid targeting peptide. The gene was expressed in .E&~rtchi~ CO/~aad therecombinant protein was isolated to homogeneity by a P affinity and anion-exchange ehromaiogra~~~. The pure proteinshowed a high thioesterase activity for oleoyl-ACP vs. o r aeyl-AGPs and therefore was identified as the coriander oleoyl-ACkthioesterase. Antibodies were raistd against the recombinant protein and used to elect the coriander thioesterase in enriched

Key wo&: Bleoyl-acyl carrier protein thioesterase; drolase; (Coriander)

acids in plants are synthesized as aqgrl-thioes-ters of acyl carrier protein (AClastids [I]. The acyl groups are tby acyyl-ACP thisesterases that

in d~~er~i~i~~ the acyl chain length. In allIyzed to date, a thioesterase specific forwas found IL?--51. Two oleoyLACP tlaio-

&erase cDNAs (clone 2-1 and clone 5-2) have beenisolated from safflower seeds [61. In addition tooleoyyl-PEP thioesterase, several thisesterases heen described that are specific for ifferent acyii-ACPs.

acyl-ACP thioesterase specific f0r laurcsyl-ACP hasn cloned from California Bay ~~~b~~~~~~~~~ cal i& w-

n& z) [7], and a medium chain- specific enzyme wasentified in Cuphea CS].The species of trae family - e.g., corian

of petroselinic acid (18: Ihoon et al. [IO] have recently shown, that this ummta

* Corresponding author. Fax: +49 0251 980 1911. Present address: Institut fiir Chemo- nnd Biosensorik, MendeE-str&e 31, 48149 Miinster, Germany.0005.2760/94/$07.00 0 1994 Elsevier Science B.V. Ali rights resewedSSDJ OOOS-2760(94)00027-P

esized as an acyB-ACP ester. la,a ~i~~~~~~t acyy-l-AGP thioesterase in toriwas characterized that was highly specific for petroseli-noyl-ACP [II].

In an attempt to done t e gene codiag fo=n: rheperroselin P this&erase frslx coriander, 2cDNA lib

esterase cDbLA done

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P. Di irm ann et al. Biochimi ca et Biophysi cs Acta 1212 (1994) 134-136 135

RLGSLTEDGLSYKEKFlVRCYEAGGTTGGGGAGCTTAACTGAAGATGGGTTGTCTTATAAAG

LIWTARMHIEIYRYPAWSDVVCTAATATGGGTC-ACTGCACGAATGCACA~G-TCTATAGATATCCTGC~~AGCGACGTGGTT

Fig. 1. DNA sequence and derived amino acid seqence of thecoriander oleoyl-ACP thioesterase. The putative start codon and thetransit peptide cleavage site are underlined.

is) MLSKGAPMPAVAAMYN(8) MATTSLASAFCSMKAVMLARDGRGWTNLCC)(S)(B)

VYPHFKTPIQCRPLTSDSISIRRRTAVSRWRSPTFSANYNGVNAaVLGVLGVLKQEQKEIEEASAKDTTFALTHSRSIGSVSIRRRYNVFLCNSS:SSRKVSPLLAVATGEQPSGVASLR:AEMK:RSSDLQLRAGNAPT:LKMING:KF:YTE:LKRLPDWSMLF:VITTIFSAAE:QWKP

CC)(S)iB)(Ci1s)(B)

KRSSSLAEKLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGGN~QSVGDKEK::GNR:::::::::::::::::"I::::::::::::I::::::::::::::::G:::PKLPQLLDDHF:L H::VFRRT:AI:S::::PDRSTSILAVM:HM::ATL:::K:::FSTDGFATTP~RKLHLIWVTARMHIEIYRYPAWSDWEIE~CQS~GRIGTRRDWIIKD::::::::: T::::::::::::::::::::::::::: I:::::V:G::KV:::::::L::ILG:::G::LE:S:RD:M::VR:T:"A"E:::T:G:T::":C:IGA_AS:NN:M:-:FL"R:

iC)(S)(Bl

FATDEVIGRATSKWVMMNQDTRRLQKVSDDVRDEYLVFCPKTPRLSFPEENNKSLKK~SKY:NG::::::::::::::E:::::::::::::E:::::::R:L::A::::::N:M:::P:CK:G:ILTIC::LS:L::TR::::STIP:E::G:IGPAFIDNVAV Km EI:K:Q:LND

(Ci(Sl(B)

LEDPAQHSRLGLSPRRADLDMNQHVNNVAYIGWVLESIPKEVLYTHELETITLDYRRECQ::::: EY:::::V:::S:::::K:::::T::::A:::::P:~ID::::QA::::::::::

ST:DYIQG::T::WN:::"::::::LK:VA::F:TV:DSIFES:HISSF::E:::::TiC)iSJ(B)(Cl(S)(B)

EWRKKSEKR:::::ps.::V

Fig. 2. Alignment of derived amino acid sequences of the oleoyl-ACPthioesterases from corainder (Cl, safflower clone 5-2 (S) 161 andCalifornia Bay 171.Amino acids that are identical with the coriandersequence are indicated by colons.

zz 400*.E5 300zE= 200*,c.s2 100

06 7 8 9 10 11 12

position of the double bondFig. 3. Substrate specificity of the coriander oleoyl-ACP thioesterase.The recombinant enzyme was purified from E. coli and thioesteraseactivity was measured with octadecenoyl-ACPs with double bonds atdifferent positions as described [ll].

postion 53 was not in frame with the rest of the codingsequence. In addition, there were stop codons in allthree reading frames before position 53. We speculate,that there may be an intron splicing mistake in theregion directly 3 of this ATG condon or a cloningartifact.

The cDNA coding for the mature part of the en-zyme was amplified by PCR and cloned into the vectorpET3d (Novagene, Madison; WI>. The cDNA was thenexpressed in Escherichiu coli BL21(DE3) and the re-combinant protein was purified to homogeneity by

IkDal

97.4 -88 -

43 -

29 -

18.4-

1234567Fig. 4. Degradation of the recombinant coriander thioesterase in acrude seed endosperm extract. Proteins were separated by SDS gelelectrophoresis and blotted onto nitrocellulose membranes. TheWestern blot was developed with anti-coriander thioesterase anti-bodies. 1, coriander thioesterase without endosperm extract; 2-6, therecombinant thioesterase was incubated with an endosperm extractfor 0 h (2) h (3), 2 h (4) and 3 h (5); 6, the extract was denatured byboiling prior to adding the recombinant thioesterase and then incu-bated for 3 h. 7, Authentic thioesterase isolated from coriander seedendosperm by alkyl-ACP affinity chromatography [8].

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c~r~~at~gra~~y as pre-to analyze the substrate

iow activity with pet

in crude endosperm extracts.em extract was ~~~~~~ed y

protein was smaller thana~t~e~t~c thioesterase.

a shorter at the ~-ter~~~~s than thesapeseed oleoyl-ACa protein in a crude extract. In order to

the question, whether the coriander CITE

to the ancubation With the recomblilant UE, T-,1.,degradation was observeled, indicating t&i ihe pro;c-.slytic activityn the mriander extract %ws Lk_Lec 2.1-t

wqime. It isnst known,ation is of any ~~ysi~%~g~

I11

[-IDl[41El[6!

[71

Qhlrogge. J.B., Jaworski, J. and Post-Beittenmiiltr. 0. 1:1993? mLipid Metabolism in Plants (Moore, T.S., ed.), pp. 3-32, CRCPress, Coca Rator, Florida.Qhlrogge. J.B., SBiae, WE. and Stu@, P.K. Ci%%l Arch.Biochem.Biophyys.89, 382-391.Heliyer, A.; Leadlay, P.F. and Slabas, AR (1992) Plant Mol.Biol. 20, 763-780.Imai, ., Nishida. I. and Murata, N. 119921 Plant Mol. Biol. Xi.139-206.

cKeon, T.A. and Stumpf 61982) J. Biol. Chem. 257, .214:-12147.Km&on, D.S., Bleibaum, J.L., Nelson, 5., &id!. J .C. antiThompson, GA. (1992) Plant Physiol. 100, 1751-!7S8.Voelker T.A., Worrel, AC., Anderscn. L..C., Hawkins, D.J., Wadke, S.E. and Davies,257, 72-74.

Weiman, R. and Spencer, G.F, (1982) J. Am. ai1 Chem Sac. 59.29-38.G&con, E.B.. Shanklin, J. and Ohkogge, J,B. (i992~ P:oc. Nat.Acad. Sci. USA 89, 11184-11188.DGrmann, P., Fre~tzen, n/a. and Ohlrogge, J.B. (i994) PlantPhysiol., in press.

2 The GenBank accession namber for the sequence reported in :hisarticle is L20978.