Planta
Planta (1993)189:425-432
9 Springer-Verlag1993
Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein Peter DOrmann L2, Friedrich Spener 2, and John B. Ohlrogge 1'* 1 Department of Botany and Plant Pathology,Michigan State University, East Lansing, MI 48824-1312, USA 2 Institut ffir Biochemie,UniversitfitMfinster, Wilhelm-Klemm-Strasse2, W-4400 Mfinster, FRG Received 11 August; accepted 23 September 1992 Abstract. Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic and lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata. Key words: Acyl carrier protein • Cuphea - Fatty acid Medium-chain - Hydrolase - Thioesterase
Introduction Several different mechanisms have been suggested for the synthesis of medium-chain fatty acids in plants. One model postulates the existence of specific acyl-acyl carrier protein (ACP) thioesterases that terminate fatty-acid elongation by the hydrolysis of a particular acyl-ACP (Stumpf 1987). Alternatively, chain length might be determined by the action of specific 3-ketoacyl-ACP synthases (condensing enzymes) or acyl-ACP acyltransferases. California bay (Umbellularia californica), a * To whomcorrespondenceshould be addressed; FAX: 1 (517) 353 1926 Abbreviations: ACP=acyl carrier protein; Ches=2-[N-cyclohexylamino]ethanesulfonic acid; DTT=dithiothreitol; MCTE= medium-chain acyl-acylcarrier protein thioesterase; Mes=2-[Nmorpholino]ethanesulfonicacid; OTE = oleoyl-acylcarrier protein thioesterase ; fatty acids are abbreviated as usual in the form: number of carbon atoms in the acyl chain ":" number of double bonds
plant that produces a lauric-acid-rich seed oil has recently been shown to contain an acyl-ACP thioesterase specific for laurate (Davies et al. 1991 ; Pollard et al. 1991). Furthermore, cloning of the California bay thioesterase cDNA and its subsequent expression in transgenic plants has resulted in lauric-acid production in seeds of Arabidopsis and Brassica (Voelker et al. 1992). In addition, in oil palm (Elaeis guineensis) mesocarp, a palmitic acidrich tissue, a high palmitoyl-ACP thioesterase activity was found (Sambanthamurthi and Oo 1990). Many species of the genus Cuphea contain unusually high levels of medium-chain fatty acids (Cs to C14) in their seed storage lipids (Graham et al. 1981; Graham 1989). Thus, Cuphea represents an alternative source for these economically important fatty acids that are usually obtained from palm kernel or coconut oils (R6bbelen 1988; Battey et al. 1989). The different species in the Cuphea genus display a diverse and facinating range of medium-chain length compositions. Furthermore, a variety of mutants in fatty-acid composition have recently been described for the species Cuphea viscosissima (Knapp 1992). The mechanism of synthesis of mediumchain fatty acids in Cuphea has not been elucidated so far. Previous studies have shown that Cuphea seeds are able to incorporate exogenous 1*C-acetate into mediumchain fatty acids (Slabas et al. 1982; Singh et al. 1986). However, in developing Cuphea lanceolata seeds, a species with high decanoic-acid levels, we could only detect a very low thioesterase activity for medium-chain acylACPs (D6rmann et al. 1991). To examine further the question of whether termination of fatty-acid elongation in developing Cuphea seeds is related to the specificity of an acyl-ACP thioesterase, as found in California bay, the activities of the acyl-ACP thioesterases in C. lanceolata and C. wrightii were investigated and the enzymes were further characterized. These two species differ in the chain length of the fatty acids found in seed triacylglycerols; C. lanceolata contains 83.2% of 10:0, whereas C. wrightii contains 29.4% of 10:0 and 53.9% of 12:0 (Graham 1989). Study of the two Cuphea species provided an opportunity to evaluate further the relation-
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
426 ship b e t w e e n a c y l - A C P thioesterase specificity a n d the c o m p o s i t i o n o f oilseeds rich in m e d i u m - c h a i n fatty acids.
Materials and methods Plant material. Cuphea lanceolata Ait. and Cuphea wrightii A. Gray plants were grown in the greenhouse under ambient light/dark conditions. Cuphea lanceolata flowers were hand-pollinated, and seeds were collected 10 15 d later. Cuphea wrightii seeds were harvested between 8 and 12 d after flowering. Seeds were frozen in liquid nitrogen and stored at - 7 0 ~ Castor bean (Ricinus communis L.) plants were grown in the greenhouse and endosperm was prepared from developing seeds.
Radioactive reagents and preparation of ac3,l-ACPs. 1-14C-Fatty acids (octanoic, decanoic, lauric, myristic, palmitic, stearic and oleic acid) with specific radioactivities of 1.94~2.15 GBq. mmol-1 were obtained from American Radiolabeled Chemicals Inc. (St. Louis, Miss., USA), Amersham (Arlington Heights, Ill., USA), New England Nuclear (Boston, Mass., USA) or Research Products International Corp. (Mount Prospect, Ill., USA). Acyl-ACPs were synthesized from Escherichia coli ACP and radioactive fatty acids using the E. coli acyl-ACP-synthetase method (Rock and Garwin 1979). Acyl-ACPs were purified by DE 52-cellulose chromatography and desalted into 10 mM 2-(N-morpholino)ethanesulfonic acid (Mes) (pH 6.1) by gel filtration on Sephadex G-25. Assay of aeyl-ACP thioesterase and protein determination. Thioesterase assays were done in total volumes of 50 ~tl. The reaction was linear up to a hydrolysis of about 20% of the substrate (data not shown); therefore, in most cases, the enzyme preparation was diluted with reaction buffer such that substrate hydrolysis was within this limit. The optimal assay conditions for the medium-chain acyl-ACP thioesterase (MCTE) and oleoyl-ACP thioesterase (OTE) markedly differed. Therefore, buffer concentration and pH, as well as the incubation time and temperature were adjusted for each enzyme. The activity of MCTE was determined with 0.8 gM decanoyl-ACP for 60min at 37 ~ in 7mM Mes-NaOH (pH 6.1), 5% (v/v) glycerol and 0.5 mM dithiothreitol (DTT). The activity of OTE was measured with 0.8 laM oleoyl-ACP for 10 min at room temperature in 35 mM 2-[N-cyclohexylamino]ethanesulfonic acid (Ches)-NaOH (pH 9.3), 5% (v/v) glycerol and 0.5 mM DTT. Reactions were stopped by adding 50 gl 1 M acetic acid in isopropanol containing 1 mM of the respective fatty acid. Free fatty acids were extracted according to Ohlrogge et al. (1978) and radioactivity determined by liquid scintillation counting. Thioesterase activity was usually expressed as pmol fatty acid hydrolyzed per min per mg protein. Protein was measured by the method of Bradford (1976). Preparation of acyl-ACP analog affinity columns. A non-hydrolyzable long-chain alkyl-ACP analog to stearoyl-ACP was prepared from 21 mg N-hexadecyliodoacetamide and 21 mg E. eoli ACP (Shanklin and Somerville 1991). This alkyl-ACP was bound to 5 ml Affi-Gel 15 (Bio-Rad, Richmond, Calif., USA). In addition, a lauroyl-ACP analog medium-chain alkyl-ACP was synthesized from 15 mg N-decyliodoacetamide and 15 mg E. coli ACP and bound to 5 ml Affi-Gel 15. N-decyliodoacetamide was prepared according to Shanklin and Somerville (1991) from 0.5 g iodoacetic anhydride and 0.2 g decylamine instead of hexadecylamine.
Protein extraction and column chromatography. All steps were carried out at 4 ~ except Mono Q and Superose 12 chromatography, which were done at room temperature. Five grams of Cuphea seeds were homogenized with a Polytron mixer in 60 ml 50 mM sodium phosphate (pH 7.5), 2 mM DTT, 5 mM EDTA and 2 mM phenylmethylsulfonyl fluoride with 6g water-insoluble polyvinylpyrrolidone. After centrifugation (30 min, 20000 9g), the supernatant was filtered over glass wool and again centrifuged (30 min, 20000"0). Protein was precipitated with 70% (w/v) ammonium
sulfate and redissolved in a minimal volume of 20 mM Tris-HC1 (pH 8.0), 2 mM DTT, 20% (v/v) glycerol. This protein extract was desalted on a Sephadex G-25 column (10 cm long, 5 cm i.d.) into 20 mM Tris-HC1 (pH 8.0), 2 mM DTT, 10% (v/v) glycerol and loaded onto a Mono Q HR 5/5 column at 0.5 ml - min- 1. After washing with the same buffer at 1 ml. min- 1, proteins were eluted with a 60-ml linear gradient from 0 to 300 mM NaCI in this buffer. For affinity chromatography, the redissolved ammoniumsulfate pellet from 20 g of seeds was desalted on Sephadex G-25 into 10 mM sodium phosphate (pH 6.8), 2 mM DTT, 10% (v/v) glycerol and diluted to 400 ml in this buffer. The protein was loaded onto the alkyl-ACP column at 0.5 ml - min 1. The column was then washed with the same buffer and protein eluted with 1 M NaC1 in this buffer. For molecular-weight estimation, the redissolved ammoniumsulfate pellet was applied at 0.5 ml ' min-1 onto a Superose 12 HR 10/30 column equilibrated with 20 mM Tris-HC1 (pH 7.5), 2 mM DTT, 5% (v/v) glycerol and 100 mM NaC1. The column was calibrated with cytochrome c (12.3 kDa), chicken albumin (43 kDa), bovine serum albumin (68 kDa) and catalase (232 kDa). Preparative gel filtration was done with a Sephadex G-75 (superfine) column (100 cm long, 2.5 cm i.d.) in 20 mM Tris-HC1 (pH 7.5), 2 mM DTT and 10% (v/v) glycerol at 8 ml 9h 1.
Gel electrophoresis and Western blot. Proteins from column fractions were separated by electrophoresis in 12% sodium dodecyl sulfate (SDS) polyacrylamide gels according to Laemmli (1970) and silver-stained (Blum et al. 1987). Alternatively, proteins were transferred onto nitrocellulose membranes and immunostained with anti-soybean OTE antibodies kindly provided by Dr. William Hitz (DuPont, Wilmington, DE, USA), as described by Post-Beittenmiller et al. (1989). Molecular weights of polypeptides were determined by comparison with the mobility of the following standard proteins: lysozyme (14.3 kDa), [3-1actoglobulin(18.4 kDa), carbonic anhydrase (29 kDa), chicken albumin (43 kDa), bovine serum albumin (68 kDa) and phosphorylase B (97.4 kDa).
Results Thioesterase activity for different acyl-ACPs in maturing Cuphea seeds. I n crude extracts of Cuphea lanceolata seeds, the thioesterase activity at p H 7.5 for o l e o y l - A C P (330 p m o l 9m i n -1 - (mg protein) -1) was a b o u t 10-fold higher t h a n for m e d i u m - c h a i n a c y l - A C P s (Fig. 1A). Alt h o u g h the p r e d o m i n a n t fatty acid in the seed lipids is decanoic acid, the thioesterase activities for l a u r o y l - A C P (48 p m o l - m i n - 1 . (mg p r o t e i n ) - 1) a n d m y r i s t o y l - A C P (46 p m o l 9 m i n 1. (mg p r o t e i n ) - 1) were higher t h a n for d e c a n o y l - A C P (28 p m o l 9m i n - 1. (mg p r o t e i n ) - 1). F u r thermore, it was s h o w n that o c t a n o y l - A C P was a p o o r thioesterase substrate. A similar a c y l - A C P hydrolysis p a t t e r n was o b t a i n e d from crude C. wrightii seed extracts with thioesterase activities for oleoyl-ACP, l a u r o y l - A C P a n d m y r i s t o y l - A C P o f 402, 32 a n d 40 p m o l " m i n -1" ( m g p r o t e i n ) -1 at p H 7.5, respectively (Fig. 1A). The thioesterase activity for m e d i u m - c h a i n a c y l - A C P s was characteristic of species a n d tissues which p r o d u c e m e d i u m - c h a i n fatty acids. As s h o w n in Fig. 1A, developing castor b e a n seeds had a m u c h lower p r o p o r t i o n o f thioesterase acitivity with m e d i u m - c h a i n a c y l - A C P substrates t h a n did Cuphea. F u r t h e r m o r e , leaf tissues o f Cuphea or other oilseed species such as d e v e l o p i n g soyb e a n seeds were also devoid o f m e d i u m - c h a i n specific a c y l - A C P thioesterase activity (data n o t shown). The
P. D 6 r m a n n et al. : Acyl-ACP thioesterases in Cuphea
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Fig. 1A, B. Chain-length specificity of acyl-ACP thioesterases in protein extracts of Cuphea seeds. Thioesterase activity was measured for 60 rain at room temperature with 0.8 p M acyl-ACP, 5% (v/v) glycerol, 0.5 m M DTT and different buffers as indicated. A Thioesterase activity for C. lanceolata (B), C. wrightii (~) and Ricinus communis (m) was determined with 7 m M sodium phosphate
(pH 7.5). Activities are given as per cent of the respective OTE activity. The OTE activity was 330, 402 and 79 pmol' min -~" (mg protein)-~ for C. lanceolata, C. wrightii and R. communis, respectively. B Cuphea lanceolata thioesterase activity was measured with 7mM Mes-NaOH, pH6.1 (N); 7mM sodium phosphate, pH 7.5 (m); and 7mM CHes-NaOH, pH 9.5 (D)
activity of the Cuphea enzyme with lauroyl-ACP was about 25% of the corresponding California bay thioesterase activity (190 pmol 9rain- ~ 9 (rag protein) ~; Davies et al. 1991). The ratio between oleoyl-ACP thioesterase and lauroyl-ACP thioesterase activity in C. lanceolata was highly dependent on pH (Fig. 1B), varying from 0.81 at pH 6.1 to 6.5 at p H 7.5 to 8.1 at pH 9.5. This indicated the existence of two thioesterases with different pH optima. The ratio between oleoyl-ACP and lauroylACP thioesterase was also variable between plants grown under different conditions. Plants grown outside were found to have M C T E activity approx. 50% of O T E activity (215, 210 and 300 pmol 9m i n - ~ 9 (rag protein)for 10: 0-ACP, 12: 0-ACP and 18 : 1-ACP, respectively). Separation ofacyl-ACP thioesterases. When an extract of C. lanceolata seeds was applied onto a M o n o Q column, two thioesterase activities were separated, a mediumchain acyl-ACP thioesterase and an oleoyl-ACP-thioes-
""
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120 80 eluted volume [ml]
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Fig. 2A, B. Separation of Cuphea acyl-ACP thioesterases by M o n o Q chromatography. Protein from 5 g seeds was loaded onto each column and eluted with a sodium chloride gradient. A Cuphea
lanceolata. B Cuphea wrightii
terase (Fig. 2A). While the OTE almost exclusively cleaved oleoyl-ACP, the M C T E had maximal activity for both lauroyl- and myristoyl-ACP (Fig. 3A). The remaining OTE activity in the M C T E fraction could be the consequence of an incomplete separation o f the O T E and M C T E by M o n o Q chromatography. In order to answer this question, M C T E and OTE fractions of a C. lanceolata seed extract were first separated by Sephadex G-75 chromatography (data not shown). After subsequent M o n o Q chromatography of the M C T E , the OTE activity was preferentially reduced in a m o u n t (Fig. 3B). Thioesterase activity of a C. wrightii seed extract could also be separated by M o n o Q chromatography into two peaks, an M C T E and an OTE activity (Fig. 2B). Again, both enzymes were analyzed for their chain-length specificity. The patterns for M C T E and OTE in C. Ianceolata and C. wrightii were very similar (Fig. 3A, C) although in the latter the activity for palmitoyl-ACP was slightly higher. p H optima and salt inhibition. The M C T E and O T E of C. lanceolata were further characterized using M o n o Qpurified enzyme fractions. The M C T E showed a broad pH optimum between 6.0 and 9.0 and was still active at pH 4.8, whereas the O T E had a sharp pH optimum at 9.3 (Fig. 4A). The two enzymes were inhibited by sodium
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
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acyI-ACP Fig. 3A-C. Chain-length specificity of acyI-ACP thioesterases in partially purified Cuphea fractions. A Cuphealanceolata MCTE and OTE preparations separated by Mono Q chromatography. B Cuphea lanceolata MCTE purified by chromatography on Sephadex G-75 and Mono Q. C Cuphea wriyhtii MCTE and OTE preparations separated by Mono Q chromatography, m, MCTE; ~, OTE.
Fig. 4A, B. pH optima and salt inhibition ofC. lanceolata acyl-ACP thioesterases. Activities were measured in Mono Q-purified MCTE and OTE preparations. A pH profiles. The following buffers were used at concentrations of 7 and 35 mM for the MCTE and OTE, respectively: sodium acetate, pH4.~5.5; Mes-NaOH, pH5.5-6.8; sodium phosphate, pH6.5-7.8; N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine(tricine)-NaOH, pH 7.5-8.8 ; ChesNaOH, pH 8.5-10.2; 3-[cyclohexylamino]-l-propanesulfonic acid(Caps)-NaOH, pH 9.7-11.2. B Inhibition of enzyme activity
chloride, but the M C T E was much more sensitive (61% activity at 20 m M NaC1) than the OTE (90% activity at 20 m M NaCI, Fig. 4B). The high sensitivity of the M C T E to sodium chloride was also observed with ammonium sulfate and all buffers tested, indicating that the inhibition is not correlated with particular ions. Both thioesterase activities could be recovered from salt solutions by desalting the enzymes using dialysis or gel filtration or by dilution. In contrast to the M C T E from California bay (Davies et al. 1991), the Cuphea M C T E did not require a particular ionic strength for maximal activity (data not shown).
preparations also contained thioesterase activity for acylCoA substrates (data not shown). Therefore, we considered the possibility that the M C T E activity might represent either an acyl-CoA thioesterase or some nonspecific hydrolase. To address this question, nonradioactive decanoyl-CoA (4 ~tM) was added to the decanoyl-ACP thioesterase assay of a crude protein extract from C. lanceolata seeds. Under these conditions (fivefold excess of decanoyl-CoA over decanoyl-ACP), the decanoyl-ACP thioesterase activity was reduced less than 35%. The thioesterase activity with and without decanoyl-CoA was linear over the incubation time of 30 min. In addition, the decanoyl-CoA thioesterase activity of the crude protein extract was determined with 4 ~M 1-a4C-decanoyl-CoA. Less then 5% of the decanoyl-CoA was hydrolyzed in 30 min (data not shown). The concentration of 4 laM was far below the critical micellar concentration of medium-chain acyl-CoAs (15-70 I.tM; Powell et al. 1981; Tippett and Neet 1982). Thus, these results indicate that the Cuphea M C T E has a strong preference for acyl-ACPs.
Competition of decanoyl-CoA and decanoyl-ACP for the MCTE. The partially purified C. lanceolata M C T E
Estimation of molecular weight. Gel filtration of a crude extract of C. lanceolata seeds on Superose 12 resulted in
Acyl-ACP thioesterase activity was measured for 60 min at room temperature with 0.8 laM acyl-ACP, 5% (v/v) glycerol, 0.5 mM DTT and 7 mM sodium phosphate (pH 7.5)
P. D 6 r m a n n et al.: AcyI-ACP thioesterases in Cuphea 9~~, "~ ~ o
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Fig. 6A, B. Affinity chromatography of C. lanceolata acyl-ACP thioesterases on non-hydrolyzable alkyl-ACP columns. A redissolved ammonium-sulfate pellet from 20 g seeds was desalted and loaded onto each column. A Long-chain alkyl-ACP column. B Medium-chain alkyl-ACP column
90% the OTE but less than 10% of the MCTE activity (Fig. 6B). The above results indicated that the OTE binding to the affinity column was more related to the ACP moiety rather than to the specific alkyl chain. At present we have no explanation for the low affinity of the MCTE for the alkyl-ACP columns, particularly considering that the California bay MCTE could be purified by ACPaffinity chromatography (Davies et al. 1991).
Purification and immunological cross-reactivity. As the MCTE did not substantially bind to any of the alkylACP columns, the enzyme was enriched 29-fold by Sephadex G-75 and Mono Q chromatography (Table 1).
Table 1. Purification of the M C T E from Cuphea lanceolata Purification step
Protein (mg)
Activity (nmol "rain -1)
Specific activity (nmol - min -x - mg -1)
Yield (%)
Crude extract a A m m o n i u m sulfate Sephadex G-75 Mono Q
48 40 9.0 0.08
3.0 2.6 2.7 0.14
0.063 0.064 0.30 1.8
100 87 90 4.7
" The crude extract was obtained from 7 g seeds
,..
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Binding to alkyl-ACP affinity columns. ACP affinity columns have previously been used to purify enzymes of plant fatty-acid metabolism (McKeon and Stumpf 1982; Hellyer and Slabas 1990; Imai et al. 1990; Davies et al. 1991 ; Thompson et al. 1991). Furthermore, a long-chain alkyl-ACP column was particularly effective in the isolation of the Ag-stearoyl-ACP desaturase from a v o c a d o (Shanklin and Somerville 1991). Therefore, we examined whether such a column is also useful to enrich thioesterases from C. lanceolata. When a seed extract was applied onto this column, almost 90% of the OTE activity, but only 10% of the MCTE was bound (Fig. 6A). Thus, the column was very effective in enriching the OTE but not the MCTE. As a further attempt to use affinity chromatography to isolate the MCTE from Cuphea, a lauroyl-ACP analog medium-chain alkyl-ACP affinity column was prepared. This column again bound about
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P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
430 Table 2. Purification of the OTE from Cuphea lanceolata Purification step
Protein (mg)
Activity ( n m o l ' m i n -1)
Crude extract a
140
87
Ammonium sulfate Long-chain alkyl-ACP column Mono Q
120 3.9 0.016
84 32 1.8
"
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1)
0.62
0.70 8.2 110
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Purification (fold)
100 97 37 2.1
1.0 1.1 13 180
The crude extract was obtained from 20 g seeds
Table 3. Kinetic data of the MCTE and OTE from Cuphea lanceolata. The MCTE was purified by Sephadex G-75 and Mono Q chromatography, and the OTE was isolated by long-chain alkylACP and Mono Q chromatography. Thioesterase activity was measured at acyl-ACP concentrations of 0.64, 0.8, 1.0, 1.28, 2.4, 4.8 and 9.6 gM, and Km and Vm,x values were obtained from Lineweaver-Burk plots
[kDa] 97.4
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F i g . 7. Gel electrophoresis and Western blot of the C. lanceolata OTE. Preparations of OTE purified by long-chain alkyl-ACP affinity and Mono Q chromatography were precipitated with 4 volumes
of ethanol and redissolved in sample buffer. Proteins in lane 1 were silver-stained; lane 2 shows a Western blot immunostained with anti-soybean OTE antibodies
This MCTE preparation contained several proteins with apparent masses of 30 to 40 kDa that cross-reacted with anti-soybean OTE antibodies in a Western blot (data not shown). However, it was not possible to confirm if these represented different isoforms of MCTE or non-specific cross-reactions of the antibody. The OTE was purified 180-fold from a crude C. lanceolata extract by chromatography on the long-chain alkyl-ACP affinity column and on Mono Q (Table 2). This fraction still contained numerous proteins as judged by polyacrylamide gel electrophoresis. On Western blots, however, only one protein band at 35.0 kDa reacted with antibodies raised against the soybean OTE (Fig. 7). Kinetic data. Apparent Km and Vmax values for the MCTE (purified by chromatography on Sephadex G-75 and Mono Q) and for OTE (purified by chromatography on a long-chain alkyl-ACP column and on Mono Q) were determined in order to further characterize the interaction between the two enzymes and different substrates (Table 3). The MCTE and OTE thioesterase preparations used for these assays were clearly separated from each other, although other proteins were present. For the MCTE, a high Km for octanoyl-ACP was found, indicat-
8: (~ACP 10:0-ACP 12:0-ACP 14:0-ACP 16:0-ACP 18:(~ACP 18: 1-ACP
Medium-chain acyl-ACP thioesterase
Oleoyl-ACP thioesterase
Km (~M)
Vmax (pmol. rain- 1)
Km Vm,x (laM) (pmol 9m i n - 1)
29.9 3.1 4.9 2.8 4.3 2.5 7.4
1.6 0.85 1.9 0.74 1.1 0.96 1.9
2.4 4.0 14.6 5.9 4.1 6.8 1.3
0.0060 0.0056 0.069 0.020 0.025 0.19 8.1
ing a low affinity for this substrate, whereas the Vmax values of all substrates were in the same range. The OTE showed the highest Vmax value for oleoyl-ACP as previously reported for the rape and California bay enzymes (Hellyer and Slabas 1990; Pollard et al. 1991). Discussion
Although it appears likely that the MCTE described in this report is responsible in large part for the production of medium-chain fatty acids in Cuphea, several aspects of the biochemistry of this system remain puzzling. In contrast to the fatty-acid distribution in the triacylglycerols, which is characterized by over 80% decanoic acid, the OTE in C. lanceolata is n~uch more active than the MCTE when assayed under optimal conditions. A partial explanation of this may relate to the pH optima of the two enzymes which are pH 6.0-9.0 for the MCTE and 9.3 for the OTE. As seed plastids lack the protonpumping activity of the photosystems of green chloroplasts, the stromal pH of seed plastids is presumably more acidic than in chloroplasts. Therefore, the OTE activity in plastids may be much lower than in chloroplasts, whereas the plastidic pH is probably still in the range of the MCTE pH optimum. The pH optimum of the Cuphea MCTE is different from that of the California bay enzyme (pH 8.5-9.9; Davies et al. 1991) which more ressembles the pH opti-
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea mum of the ubiquitous OTE. In addition, the Cuphea M C T E does not bind to ACP affinity columns, whereas the bay M C T E , as well as the OTEs studied so far, do. The broad acyl-ACP specificity o f the Cuphea M C T E also differs from the bay M C T E and from the OTE. The latter two enzymes show a sharp specificity for only one substrate. These differences may indicate that the phylogenetic relationship between the OTE and the bay M C T E is closer than between the OTE and the Cuphea MCTE. Both Cuphea and California bay M C T E in-vitro activities are lower than the in-vivo rate o f accumulation of medium-chain fatty acids in seed lipids. F r o m our data, an in-vitro decanoyl-ACP thioesterase activity of 1.25 pmol 9min-~ 9 (embryo)-1 was obtained for C. lanceolata which is much less than the in-vivo incorporation of decanoic acid into seed triacylglycerols (83 pmol 9m i n - 1. (embryo)- ~; calculated from data of Bafor et al. 1990). A similar discrepancy can be calculated between the in-vitro lauroyl-ACP thioesterase activity (2.7 n m o i . m i n - 1. (cotyledon pair)- 1) and the in-vivo accumulation of lauric acid (56 n m o l - r a i n 1. (cotyledon pair)-1) in California bay seeds (calculated from data of Davies et al. 1991). These comparisons indicate that the in-vitro assay of M C T E in some way substantially underestimates the in-vivo activity. Possibly, the M C T E requires close association with the fattyacid synthase enzymes or other factors for optimal activity. In this regard, the purification of the Cuphea M C T E to homogeneity may not lead to results that have a better relation to in-vivo activities than the data obtained in this work. The Cuphea M C T E is most active for lauroyl- and myristoyl-ACP, and shows less activity for decanoyl-, palmitoyl-, stearoyl- and oleoyl-ACP. Presumably the binding site of the Cuphea M C T E has optimal affinity for acyl-ACPs with chain lengths of 10-14 carbon atoms whereas acyl-ACPs with 8 or less acyl carbon atoms are excluded. Decanoyl-ACP is therefore the first product of the acyl-ACP elongation by the fatty-acid synthase that will be hydrolyzed by the Cuphea MCTE. In Cuphea seeds the pools of 12:0-ACP, 14:0-ACP, 16:0-ACP and 18:0-ACp are very low (Singh et al. 1986), such that the 12:0-ACP and 14:0-ACP thioesterase activities of the M C T E cannot produce appreciable amounts of the respective free fatty acids. After elongation and desaturation to 18 : 1-ACP, the relatively high activity of the OTE may be essential to ensure that all 18:I-ACP will be hydrolyzed. It is puzzling that the chain-length specificity of the C. lanceolata and C. wrightii M C T E is almost identical, although the seed triacylglycerols of C. wrightii contain considerable amounts of lauric acid in additon to decanoic acid. Thus, these results emphasize the incomplete state of our understanding regarding the mechanistic details leading to the final fatty acid composition of seed storage triacylglycerols. The determination of fatty-acid chain length can be controlled by a variety of mechanisms. The condensing enzymes of most known soluble fatty-acid synthases show decreased rates of elongation when the acyl primer
431 reaches a length of either 16 or 18 carbons. In addition to condensing-enzyme specificity, in Saccharomyces cerevisiae, chain termination is accomplished when the length of acyl groups attached to the multifunctional fatty-acid synthase reaches 16 carbons and it is transferred from ACP to Coenzyme A by palmitoyl-transferase. In animals, termination o f fatty-acid synthesis is brought on by hydrolysis o f the acyl chain from the synthase by either a palmitoyl-specific thioesterase or in the case of mammary glands, by a medium-chain-specific enzyme. In plants, in addition to chain-length-specific acyl-ACP thioesterases, transfer o f fatty acids from ACP to glycerol-3-phosphate also serves as a termination reaction. In this study we have demonstrated that C. lanceolata and C. wrightii seeds contain at least two acylACP thioesterases, with one specific for oleoyl-ACP and one for medium-chain acyl-ACPs. In this regard, Cuphea is similar to California bay and, therefore, these results indicate that these two phylogenetically distinct species utilize a similar mechanism for the production of medium-chain fatty acids in seeds. This work was supported by grants of the German Federal Ministry for Research and Technology (F6rderkennzeichen 0319412 C), by the German Academic Exchange Service and by the National Science Foundation (grant number DCB 9005290). The authors would like to thank Dr. William Hitz, DuPont, for kindly providing anti-soybean OTE antibodies. Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.
References
Bafor, M., Jonsson, L., Stobart, A.K., Stymne, S. (1990) Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata. Biochem. J. 272, 31-38 Battey, J.F., Schmid, K.M., Ohlrogge, J.B. (1989) Genetic engineering for plant oils: Potential and limitations. Trends Biotechnol. 7, 122-125 Blum, H., Beier, H., Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Davies, H.M., Anderson, L., Fan, C., Hawkins, D.J. (1991) Developmental induction, purification, and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbellularia californica. Arch. Biochem. Biophys. 290, 37-45 D6rmann, P., Schmid, P.C., Robers, M., Spener, F. (1991) AcylACP thioesterase(s) for the cleavage of medium- and long-chain acyl-ACPs in Cuphea lanceolata seeds. (Abstr.) Biol. Chem. Hoppe-Seyler 372, 528-529 Graham, S.A. (1989) Cuphea:A new plant source of medium-chain fatty acids. Crit. Rev. Food Sci. Nutr. 28, 139-173 Graham, S.A., Hirsinger, F., R6bbelen, G. (1981) Fatty acids of Cuphea (Lythraceae) seed lipids and their systematic significance. Am. J. Bot. 68, 908-917 Hellyer, A., Slabas, A. (1990) Acyl-[acylcarrier protein] thioesterase from oil seed rape: Purification and characterization. In: Plant lipid biochemistry, structure and utilization, pp. 157-159, Quinn, P.J., Harwood, J.L., eds. Portland Press, London Imai, H., Nishida, I., Murata, N. (1990) Acyl-(acyl-carrier protein) hydrolase of squash cotyledons: Purification and characterization. In: Plant lipid biochemistry, structure and utilization, pp.
432 160-162, Quinn, P.J., Harwood, J.L., eds. Portland Press, London Knapp, S.J. (1992) Modifying the seed oils of Cuphea - a new commercial source of caprylic, capric, lauric, and myristic acid. J. Am. Oil. Chem. Soc. (in press) Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 McKeon, T.A., Stumpf, P.K. (1982) Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acylacyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem. 257, 12141-12147 Ohlrogge, J.B., Shine, W.E., Stumpf, P.K. (1978) Fat metabolism in higher plants. Characterization of plant acyl-ACP and acylCoA hydrolases. Arch. Biochem. Biophys. 189, 382-391 Pollard, M.R., Anderson, L , Fan, C., Hawkins, D.J., Davies, H.M. (1991) A specific aeyI-ACP thioesterase implicated in mediumchain fatty acid production in immature cotyledons of Umbellularia caliJbrnia. Arch. Biochem. Biophys. 284, 306-312 Post-Beittenmiller, M.A., Schmid, K.M., Ohlrogge, J.B. (1989) Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants. Plant Cell 1, 889-899 Powell, G.L., Grothusen, J.R., Zimmerman, J.K., Evans, C.A., Fish, W.W. (1981) A re-examination of some properties of fatty acyl-CoA micelles. J. Biol. Chem. 256, 12740-12747 R6bbelen, G. (1988) Development of new industrial oil crops. In: Proceedings of the world conference on biotechnology for the fats and oils industry, pp. 78-86, Applewhite, T.H., ed. American Oil Chemists' Society, Champaign, Illinois Rock, C.O., Garwin, J.L. (1979) Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J. Biol. Chem. 254, 7123-7128
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea Sambanthamurthi, R., Oo, K.-C. (1990) Thioesterase activity in the oil palm (Elaeis 9uineensis) mesocarp. In: Plant lipid biochemistry, structure and utilization, pp. 166-168, Quinn, P.J., Hatwood, J.L., eds. Portland Press, London Shanklin, J., Somerville, C. (1991) Stearoyl-acyl-earrier-proteindesaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proc. Natl. Acad. Sci. USA 88, 2510-2514 Singh, S.S., Nee, T.Y., Pollard, M.R. (1986) Acetate and mevalonate labeling studies with developing Cuphea lutea seeds. Lipids, 21, 143 149 Slabas, A.R., Roberts, P.A., Ormesher, J., Hammond, E.W. (1982) Cuphea procumbens. A model system for studying the mechanism of medium-chain fatty acid biosynthesis in plants. Biochem. Biophys. Acta 711,411~420 Stumpf, P.K. (1987) The biosynthesis of saturated fatty acids. In: The biochemistry of plants, vol. 9, Lipids: Structure and function, pp. 121-136, Stumpf, P.K., ed. Academic Press, New York Thompson, G.A., Scherer, D.E., Foxall-Van Aken, S., Kenny, J.W., Young, H.L, Shintani, D.K., Kridl, J.C., Knauf, V.C. (1991) Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity. Proc. Natl. Acad. Sci. USA 88, 2578-2582 Tippett, P.S., Neet, K.E. (1982) Specific inhibition of glucokinase by long chain acyl Coenzyme A below the critical micelle concentration. J. Biol. Chem. 257, 12839-12845 Voelker, T.A., Worrell, A.C., Anderson, L., Bleibaum, J., Fan, C., Hawkins, D.J., Radke, S.E., Davies, H.M. (1992) Fatty acid biosynthesis redirected to medium-chains in transgenic oilseed plants. Science 257, 72-74
Planta (1993)189:425-432
9 Springer-Verlag1993
Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein Peter DOrmann L2, Friedrich Spener 2, and John B. Ohlrogge 1'* 1 Department of Botany and Plant Pathology,Michigan State University, East Lansing, MI 48824-1312, USA 2 Institut ffir Biochemie,UniversitfitMfinster, Wilhelm-Klemm-Strasse2, W-4400 Mfinster, FRG Received 11 August; accepted 23 September 1992 Abstract. Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic and lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata. Key words: Acyl carrier protein • Cuphea - Fatty acid Medium-chain - Hydrolase - Thioesterase
Introduction Several different mechanisms have been suggested for the synthesis of medium-chain fatty acids in plants. One model postulates the existence of specific acyl-acyl carrier protein (ACP) thioesterases that terminate fatty-acid elongation by the hydrolysis of a particular acyl-ACP (Stumpf 1987). Alternatively, chain length might be determined by the action of specific 3-ketoacyl-ACP synthases (condensing enzymes) or acyl-ACP acyltransferases. California bay (Umbellularia californica), a * To whomcorrespondenceshould be addressed; FAX: 1 (517) 353 1926 Abbreviations: ACP=acyl carrier protein; Ches=2-[N-cyclohexylamino]ethanesulfonic acid; DTT=dithiothreitol; MCTE= medium-chain acyl-acylcarrier protein thioesterase; Mes=2-[Nmorpholino]ethanesulfonicacid; OTE = oleoyl-acylcarrier protein thioesterase ; fatty acids are abbreviated as usual in the form: number of carbon atoms in the acyl chain ":" number of double bonds
plant that produces a lauric-acid-rich seed oil has recently been shown to contain an acyl-ACP thioesterase specific for laurate (Davies et al. 1991 ; Pollard et al. 1991). Furthermore, cloning of the California bay thioesterase cDNA and its subsequent expression in transgenic plants has resulted in lauric-acid production in seeds of Arabidopsis and Brassica (Voelker et al. 1992). In addition, in oil palm (Elaeis guineensis) mesocarp, a palmitic acidrich tissue, a high palmitoyl-ACP thioesterase activity was found (Sambanthamurthi and Oo 1990). Many species of the genus Cuphea contain unusually high levels of medium-chain fatty acids (Cs to C14) in their seed storage lipids (Graham et al. 1981; Graham 1989). Thus, Cuphea represents an alternative source for these economically important fatty acids that are usually obtained from palm kernel or coconut oils (R6bbelen 1988; Battey et al. 1989). The different species in the Cuphea genus display a diverse and facinating range of medium-chain length compositions. Furthermore, a variety of mutants in fatty-acid composition have recently been described for the species Cuphea viscosissima (Knapp 1992). The mechanism of synthesis of mediumchain fatty acids in Cuphea has not been elucidated so far. Previous studies have shown that Cuphea seeds are able to incorporate exogenous 1*C-acetate into mediumchain fatty acids (Slabas et al. 1982; Singh et al. 1986). However, in developing Cuphea lanceolata seeds, a species with high decanoic-acid levels, we could only detect a very low thioesterase activity for medium-chain acylACPs (D6rmann et al. 1991). To examine further the question of whether termination of fatty-acid elongation in developing Cuphea seeds is related to the specificity of an acyl-ACP thioesterase, as found in California bay, the activities of the acyl-ACP thioesterases in C. lanceolata and C. wrightii were investigated and the enzymes were further characterized. These two species differ in the chain length of the fatty acids found in seed triacylglycerols; C. lanceolata contains 83.2% of 10:0, whereas C. wrightii contains 29.4% of 10:0 and 53.9% of 12:0 (Graham 1989). Study of the two Cuphea species provided an opportunity to evaluate further the relation-
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
426 ship b e t w e e n a c y l - A C P thioesterase specificity a n d the c o m p o s i t i o n o f oilseeds rich in m e d i u m - c h a i n fatty acids.
Materials and methods Plant material. Cuphea lanceolata Ait. and Cuphea wrightii A. Gray plants were grown in the greenhouse under ambient light/dark conditions. Cuphea lanceolata flowers were hand-pollinated, and seeds were collected 10 15 d later. Cuphea wrightii seeds were harvested between 8 and 12 d after flowering. Seeds were frozen in liquid nitrogen and stored at - 7 0 ~ Castor bean (Ricinus communis L.) plants were grown in the greenhouse and endosperm was prepared from developing seeds.
Radioactive reagents and preparation of ac3,l-ACPs. 1-14C-Fatty acids (octanoic, decanoic, lauric, myristic, palmitic, stearic and oleic acid) with specific radioactivities of 1.94~2.15 GBq. mmol-1 were obtained from American Radiolabeled Chemicals Inc. (St. Louis, Miss., USA), Amersham (Arlington Heights, Ill., USA), New England Nuclear (Boston, Mass., USA) or Research Products International Corp. (Mount Prospect, Ill., USA). Acyl-ACPs were synthesized from Escherichia coli ACP and radioactive fatty acids using the E. coli acyl-ACP-synthetase method (Rock and Garwin 1979). Acyl-ACPs were purified by DE 52-cellulose chromatography and desalted into 10 mM 2-(N-morpholino)ethanesulfonic acid (Mes) (pH 6.1) by gel filtration on Sephadex G-25. Assay of aeyl-ACP thioesterase and protein determination. Thioesterase assays were done in total volumes of 50 ~tl. The reaction was linear up to a hydrolysis of about 20% of the substrate (data not shown); therefore, in most cases, the enzyme preparation was diluted with reaction buffer such that substrate hydrolysis was within this limit. The optimal assay conditions for the medium-chain acyl-ACP thioesterase (MCTE) and oleoyl-ACP thioesterase (OTE) markedly differed. Therefore, buffer concentration and pH, as well as the incubation time and temperature were adjusted for each enzyme. The activity of MCTE was determined with 0.8 gM decanoyl-ACP for 60min at 37 ~ in 7mM Mes-NaOH (pH 6.1), 5% (v/v) glycerol and 0.5 mM dithiothreitol (DTT). The activity of OTE was measured with 0.8 laM oleoyl-ACP for 10 min at room temperature in 35 mM 2-[N-cyclohexylamino]ethanesulfonic acid (Ches)-NaOH (pH 9.3), 5% (v/v) glycerol and 0.5 mM DTT. Reactions were stopped by adding 50 gl 1 M acetic acid in isopropanol containing 1 mM of the respective fatty acid. Free fatty acids were extracted according to Ohlrogge et al. (1978) and radioactivity determined by liquid scintillation counting. Thioesterase activity was usually expressed as pmol fatty acid hydrolyzed per min per mg protein. Protein was measured by the method of Bradford (1976). Preparation of acyl-ACP analog affinity columns. A non-hydrolyzable long-chain alkyl-ACP analog to stearoyl-ACP was prepared from 21 mg N-hexadecyliodoacetamide and 21 mg E. eoli ACP (Shanklin and Somerville 1991). This alkyl-ACP was bound to 5 ml Affi-Gel 15 (Bio-Rad, Richmond, Calif., USA). In addition, a lauroyl-ACP analog medium-chain alkyl-ACP was synthesized from 15 mg N-decyliodoacetamide and 15 mg E. coli ACP and bound to 5 ml Affi-Gel 15. N-decyliodoacetamide was prepared according to Shanklin and Somerville (1991) from 0.5 g iodoacetic anhydride and 0.2 g decylamine instead of hexadecylamine.
Protein extraction and column chromatography. All steps were carried out at 4 ~ except Mono Q and Superose 12 chromatography, which were done at room temperature. Five grams of Cuphea seeds were homogenized with a Polytron mixer in 60 ml 50 mM sodium phosphate (pH 7.5), 2 mM DTT, 5 mM EDTA and 2 mM phenylmethylsulfonyl fluoride with 6g water-insoluble polyvinylpyrrolidone. After centrifugation (30 min, 20000 9g), the supernatant was filtered over glass wool and again centrifuged (30 min, 20000"0). Protein was precipitated with 70% (w/v) ammonium
sulfate and redissolved in a minimal volume of 20 mM Tris-HC1 (pH 8.0), 2 mM DTT, 20% (v/v) glycerol. This protein extract was desalted on a Sephadex G-25 column (10 cm long, 5 cm i.d.) into 20 mM Tris-HC1 (pH 8.0), 2 mM DTT, 10% (v/v) glycerol and loaded onto a Mono Q HR 5/5 column at 0.5 ml - min- 1. After washing with the same buffer at 1 ml. min- 1, proteins were eluted with a 60-ml linear gradient from 0 to 300 mM NaCI in this buffer. For affinity chromatography, the redissolved ammoniumsulfate pellet from 20 g of seeds was desalted on Sephadex G-25 into 10 mM sodium phosphate (pH 6.8), 2 mM DTT, 10% (v/v) glycerol and diluted to 400 ml in this buffer. The protein was loaded onto the alkyl-ACP column at 0.5 ml - min 1. The column was then washed with the same buffer and protein eluted with 1 M NaC1 in this buffer. For molecular-weight estimation, the redissolved ammoniumsulfate pellet was applied at 0.5 ml ' min-1 onto a Superose 12 HR 10/30 column equilibrated with 20 mM Tris-HC1 (pH 7.5), 2 mM DTT, 5% (v/v) glycerol and 100 mM NaC1. The column was calibrated with cytochrome c (12.3 kDa), chicken albumin (43 kDa), bovine serum albumin (68 kDa) and catalase (232 kDa). Preparative gel filtration was done with a Sephadex G-75 (superfine) column (100 cm long, 2.5 cm i.d.) in 20 mM Tris-HC1 (pH 7.5), 2 mM DTT and 10% (v/v) glycerol at 8 ml 9h 1.
Gel electrophoresis and Western blot. Proteins from column fractions were separated by electrophoresis in 12% sodium dodecyl sulfate (SDS) polyacrylamide gels according to Laemmli (1970) and silver-stained (Blum et al. 1987). Alternatively, proteins were transferred onto nitrocellulose membranes and immunostained with anti-soybean OTE antibodies kindly provided by Dr. William Hitz (DuPont, Wilmington, DE, USA), as described by Post-Beittenmiller et al. (1989). Molecular weights of polypeptides were determined by comparison with the mobility of the following standard proteins: lysozyme (14.3 kDa), [3-1actoglobulin(18.4 kDa), carbonic anhydrase (29 kDa), chicken albumin (43 kDa), bovine serum albumin (68 kDa) and phosphorylase B (97.4 kDa).
Results Thioesterase activity for different acyl-ACPs in maturing Cuphea seeds. I n crude extracts of Cuphea lanceolata seeds, the thioesterase activity at p H 7.5 for o l e o y l - A C P (330 p m o l 9m i n -1 - (mg protein) -1) was a b o u t 10-fold higher t h a n for m e d i u m - c h a i n a c y l - A C P s (Fig. 1A). Alt h o u g h the p r e d o m i n a n t fatty acid in the seed lipids is decanoic acid, the thioesterase activities for l a u r o y l - A C P (48 p m o l - m i n - 1 . (mg p r o t e i n ) - 1) a n d m y r i s t o y l - A C P (46 p m o l 9 m i n 1. (mg p r o t e i n ) - 1) were higher t h a n for d e c a n o y l - A C P (28 p m o l 9m i n - 1. (mg p r o t e i n ) - 1). F u r thermore, it was s h o w n that o c t a n o y l - A C P was a p o o r thioesterase substrate. A similar a c y l - A C P hydrolysis p a t t e r n was o b t a i n e d from crude C. wrightii seed extracts with thioesterase activities for oleoyl-ACP, l a u r o y l - A C P a n d m y r i s t o y l - A C P o f 402, 32 a n d 40 p m o l " m i n -1" ( m g p r o t e i n ) -1 at p H 7.5, respectively (Fig. 1A). The thioesterase activity for m e d i u m - c h a i n a c y l - A C P s was characteristic of species a n d tissues which p r o d u c e m e d i u m - c h a i n fatty acids. As s h o w n in Fig. 1A, developing castor b e a n seeds had a m u c h lower p r o p o r t i o n o f thioesterase acitivity with m e d i u m - c h a i n a c y l - A C P substrates t h a n did Cuphea. F u r t h e r m o r e , leaf tissues o f Cuphea or other oilseed species such as d e v e l o p i n g soyb e a n seeds were also devoid o f m e d i u m - c h a i n specific a c y l - A C P thioesterase activity (data n o t shown). The
P. D 6 r m a n n et al. : Acyl-ACP thioesterases in Cuphea
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Fig. 1A, B. Chain-length specificity of acyl-ACP thioesterases in protein extracts of Cuphea seeds. Thioesterase activity was measured for 60 rain at room temperature with 0.8 p M acyl-ACP, 5% (v/v) glycerol, 0.5 m M DTT and different buffers as indicated. A Thioesterase activity for C. lanceolata (B), C. wrightii (~) and Ricinus communis (m) was determined with 7 m M sodium phosphate
(pH 7.5). Activities are given as per cent of the respective OTE activity. The OTE activity was 330, 402 and 79 pmol' min -~" (mg protein)-~ for C. lanceolata, C. wrightii and R. communis, respectively. B Cuphea lanceolata thioesterase activity was measured with 7mM Mes-NaOH, pH6.1 (N); 7mM sodium phosphate, pH 7.5 (m); and 7mM CHes-NaOH, pH 9.5 (D)
activity of the Cuphea enzyme with lauroyl-ACP was about 25% of the corresponding California bay thioesterase activity (190 pmol 9rain- ~ 9 (rag protein) ~; Davies et al. 1991). The ratio between oleoyl-ACP thioesterase and lauroyl-ACP thioesterase activity in C. lanceolata was highly dependent on pH (Fig. 1B), varying from 0.81 at pH 6.1 to 6.5 at p H 7.5 to 8.1 at pH 9.5. This indicated the existence of two thioesterases with different pH optima. The ratio between oleoyl-ACP and lauroylACP thioesterase was also variable between plants grown under different conditions. Plants grown outside were found to have M C T E activity approx. 50% of O T E activity (215, 210 and 300 pmol 9m i n - ~ 9 (rag protein)for 10: 0-ACP, 12: 0-ACP and 18 : 1-ACP, respectively). Separation ofacyl-ACP thioesterases. When an extract of C. lanceolata seeds was applied onto a M o n o Q column, two thioesterase activities were separated, a mediumchain acyl-ACP thioesterase and an oleoyl-ACP-thioes-
""
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lanceolata. B Cuphea wrightii
terase (Fig. 2A). While the OTE almost exclusively cleaved oleoyl-ACP, the M C T E had maximal activity for both lauroyl- and myristoyl-ACP (Fig. 3A). The remaining OTE activity in the M C T E fraction could be the consequence of an incomplete separation o f the O T E and M C T E by M o n o Q chromatography. In order to answer this question, M C T E and OTE fractions of a C. lanceolata seed extract were first separated by Sephadex G-75 chromatography (data not shown). After subsequent M o n o Q chromatography of the M C T E , the OTE activity was preferentially reduced in a m o u n t (Fig. 3B). Thioesterase activity of a C. wrightii seed extract could also be separated by M o n o Q chromatography into two peaks, an M C T E and an OTE activity (Fig. 2B). Again, both enzymes were analyzed for their chain-length specificity. The patterns for M C T E and OTE in C. Ianceolata and C. wrightii were very similar (Fig. 3A, C) although in the latter the activity for palmitoyl-ACP was slightly higher. p H optima and salt inhibition. The M C T E and O T E of C. lanceolata were further characterized using M o n o Qpurified enzyme fractions. The M C T E showed a broad pH optimum between 6.0 and 9.0 and was still active at pH 4.8, whereas the O T E had a sharp pH optimum at 9.3 (Fig. 4A). The two enzymes were inhibited by sodium
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
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acyI-ACP Fig. 3A-C. Chain-length specificity of acyI-ACP thioesterases in partially purified Cuphea fractions. A Cuphealanceolata MCTE and OTE preparations separated by Mono Q chromatography. B Cuphea lanceolata MCTE purified by chromatography on Sephadex G-75 and Mono Q. C Cuphea wriyhtii MCTE and OTE preparations separated by Mono Q chromatography, m, MCTE; ~, OTE.
Fig. 4A, B. pH optima and salt inhibition ofC. lanceolata acyl-ACP thioesterases. Activities were measured in Mono Q-purified MCTE and OTE preparations. A pH profiles. The following buffers were used at concentrations of 7 and 35 mM for the MCTE and OTE, respectively: sodium acetate, pH4.~5.5; Mes-NaOH, pH5.5-6.8; sodium phosphate, pH6.5-7.8; N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine(tricine)-NaOH, pH 7.5-8.8 ; ChesNaOH, pH 8.5-10.2; 3-[cyclohexylamino]-l-propanesulfonic acid(Caps)-NaOH, pH 9.7-11.2. B Inhibition of enzyme activity
chloride, but the M C T E was much more sensitive (61% activity at 20 m M NaC1) than the OTE (90% activity at 20 m M NaCI, Fig. 4B). The high sensitivity of the M C T E to sodium chloride was also observed with ammonium sulfate and all buffers tested, indicating that the inhibition is not correlated with particular ions. Both thioesterase activities could be recovered from salt solutions by desalting the enzymes using dialysis or gel filtration or by dilution. In contrast to the M C T E from California bay (Davies et al. 1991), the Cuphea M C T E did not require a particular ionic strength for maximal activity (data not shown).
preparations also contained thioesterase activity for acylCoA substrates (data not shown). Therefore, we considered the possibility that the M C T E activity might represent either an acyl-CoA thioesterase or some nonspecific hydrolase. To address this question, nonradioactive decanoyl-CoA (4 ~tM) was added to the decanoyl-ACP thioesterase assay of a crude protein extract from C. lanceolata seeds. Under these conditions (fivefold excess of decanoyl-CoA over decanoyl-ACP), the decanoyl-ACP thioesterase activity was reduced less than 35%. The thioesterase activity with and without decanoyl-CoA was linear over the incubation time of 30 min. In addition, the decanoyl-CoA thioesterase activity of the crude protein extract was determined with 4 ~M 1-a4C-decanoyl-CoA. Less then 5% of the decanoyl-CoA was hydrolyzed in 30 min (data not shown). The concentration of 4 laM was far below the critical micellar concentration of medium-chain acyl-CoAs (15-70 I.tM; Powell et al. 1981; Tippett and Neet 1982). Thus, these results indicate that the Cuphea M C T E has a strong preference for acyl-ACPs.
Competition of decanoyl-CoA and decanoyl-ACP for the MCTE. The partially purified C. lanceolata M C T E
Estimation of molecular weight. Gel filtration of a crude extract of C. lanceolata seeds on Superose 12 resulted in
Acyl-ACP thioesterase activity was measured for 60 min at room temperature with 0.8 laM acyl-ACP, 5% (v/v) glycerol, 0.5 mM DTT and 7 mM sodium phosphate (pH 7.5)
P. D 6 r m a n n et al.: AcyI-ACP thioesterases in Cuphea 9~~, "~ ~ o
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Fig. 6A, B. Affinity chromatography of C. lanceolata acyl-ACP thioesterases on non-hydrolyzable alkyl-ACP columns. A redissolved ammonium-sulfate pellet from 20 g seeds was desalted and loaded onto each column. A Long-chain alkyl-ACP column. B Medium-chain alkyl-ACP column
90% the OTE but less than 10% of the MCTE activity (Fig. 6B). The above results indicated that the OTE binding to the affinity column was more related to the ACP moiety rather than to the specific alkyl chain. At present we have no explanation for the low affinity of the MCTE for the alkyl-ACP columns, particularly considering that the California bay MCTE could be purified by ACPaffinity chromatography (Davies et al. 1991).
Purification and immunological cross-reactivity. As the MCTE did not substantially bind to any of the alkylACP columns, the enzyme was enriched 29-fold by Sephadex G-75 and Mono Q chromatography (Table 1).
Table 1. Purification of the M C T E from Cuphea lanceolata Purification step
Protein (mg)
Activity (nmol "rain -1)
Specific activity (nmol - min -x - mg -1)
Yield (%)
Crude extract a A m m o n i u m sulfate Sephadex G-75 Mono Q
48 40 9.0 0.08
3.0 2.6 2.7 0.14
0.063 0.064 0.30 1.8
100 87 90 4.7
" The crude extract was obtained from 7 g seeds
,..
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Binding to alkyl-ACP affinity columns. ACP affinity columns have previously been used to purify enzymes of plant fatty-acid metabolism (McKeon and Stumpf 1982; Hellyer and Slabas 1990; Imai et al. 1990; Davies et al. 1991 ; Thompson et al. 1991). Furthermore, a long-chain alkyl-ACP column was particularly effective in the isolation of the Ag-stearoyl-ACP desaturase from a v o c a d o (Shanklin and Somerville 1991). Therefore, we examined whether such a column is also useful to enrich thioesterases from C. lanceolata. When a seed extract was applied onto this column, almost 90% of the OTE activity, but only 10% of the MCTE was bound (Fig. 6A). Thus, the column was very effective in enriching the OTE but not the MCTE. As a further attempt to use affinity chromatography to isolate the MCTE from Cuphea, a lauroyl-ACP analog medium-chain alkyl-ACP affinity column was prepared. This column again bound about
1
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P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea
430 Table 2. Purification of the OTE from Cuphea lanceolata Purification step
Protein (mg)
Activity ( n m o l ' m i n -1)
Crude extract a
140
87
Ammonium sulfate Long-chain alkyl-ACP column Mono Q
120 3.9 0.016
84 32 1.8
"
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1)
0.62
0.70 8.2 110
Yield (%)
Purification (fold)
100 97 37 2.1
1.0 1.1 13 180
The crude extract was obtained from 20 g seeds
Table 3. Kinetic data of the MCTE and OTE from Cuphea lanceolata. The MCTE was purified by Sephadex G-75 and Mono Q chromatography, and the OTE was isolated by long-chain alkylACP and Mono Q chromatography. Thioesterase activity was measured at acyl-ACP concentrations of 0.64, 0.8, 1.0, 1.28, 2.4, 4.8 and 9.6 gM, and Km and Vm,x values were obtained from Lineweaver-Burk plots
[kDa] 97.4
-
68
-
43
-
29
-
18.4
-
14.3
-
Substrate
1
2
F i g . 7. Gel electrophoresis and Western blot of the C. lanceolata OTE. Preparations of OTE purified by long-chain alkyl-ACP affinity and Mono Q chromatography were precipitated with 4 volumes
of ethanol and redissolved in sample buffer. Proteins in lane 1 were silver-stained; lane 2 shows a Western blot immunostained with anti-soybean OTE antibodies
This MCTE preparation contained several proteins with apparent masses of 30 to 40 kDa that cross-reacted with anti-soybean OTE antibodies in a Western blot (data not shown). However, it was not possible to confirm if these represented different isoforms of MCTE or non-specific cross-reactions of the antibody. The OTE was purified 180-fold from a crude C. lanceolata extract by chromatography on the long-chain alkyl-ACP affinity column and on Mono Q (Table 2). This fraction still contained numerous proteins as judged by polyacrylamide gel electrophoresis. On Western blots, however, only one protein band at 35.0 kDa reacted with antibodies raised against the soybean OTE (Fig. 7). Kinetic data. Apparent Km and Vmax values for the MCTE (purified by chromatography on Sephadex G-75 and Mono Q) and for OTE (purified by chromatography on a long-chain alkyl-ACP column and on Mono Q) were determined in order to further characterize the interaction between the two enzymes and different substrates (Table 3). The MCTE and OTE thioesterase preparations used for these assays were clearly separated from each other, although other proteins were present. For the MCTE, a high Km for octanoyl-ACP was found, indicat-
8: (~ACP 10:0-ACP 12:0-ACP 14:0-ACP 16:0-ACP 18:(~ACP 18: 1-ACP
Medium-chain acyl-ACP thioesterase
Oleoyl-ACP thioesterase
Km (~M)
Vmax (pmol. rain- 1)
Km Vm,x (laM) (pmol 9m i n - 1)
29.9 3.1 4.9 2.8 4.3 2.5 7.4
1.6 0.85 1.9 0.74 1.1 0.96 1.9
2.4 4.0 14.6 5.9 4.1 6.8 1.3
0.0060 0.0056 0.069 0.020 0.025 0.19 8.1
ing a low affinity for this substrate, whereas the Vmax values of all substrates were in the same range. The OTE showed the highest Vmax value for oleoyl-ACP as previously reported for the rape and California bay enzymes (Hellyer and Slabas 1990; Pollard et al. 1991). Discussion
Although it appears likely that the MCTE described in this report is responsible in large part for the production of medium-chain fatty acids in Cuphea, several aspects of the biochemistry of this system remain puzzling. In contrast to the fatty-acid distribution in the triacylglycerols, which is characterized by over 80% decanoic acid, the OTE in C. lanceolata is n~uch more active than the MCTE when assayed under optimal conditions. A partial explanation of this may relate to the pH optima of the two enzymes which are pH 6.0-9.0 for the MCTE and 9.3 for the OTE. As seed plastids lack the protonpumping activity of the photosystems of green chloroplasts, the stromal pH of seed plastids is presumably more acidic than in chloroplasts. Therefore, the OTE activity in plastids may be much lower than in chloroplasts, whereas the plastidic pH is probably still in the range of the MCTE pH optimum. The pH optimum of the Cuphea MCTE is different from that of the California bay enzyme (pH 8.5-9.9; Davies et al. 1991) which more ressembles the pH opti-
P. D6rmann et al.: Acyl-ACP thioesterases in Cuphea mum of the ubiquitous OTE. In addition, the Cuphea M C T E does not bind to ACP affinity columns, whereas the bay M C T E , as well as the OTEs studied so far, do. The broad acyl-ACP specificity o f the Cuphea M C T E also differs from the bay M C T E and from the OTE. The latter two enzymes show a sharp specificity for only one substrate. These differences may indicate that the phylogenetic relationship between the OTE and the bay M C T E is closer than between the OTE and the Cuphea MCTE. Both Cuphea and California bay M C T E in-vitro activities are lower than the in-vivo rate o f accumulation of medium-chain fatty acids in seed lipids. F r o m our data, an in-vitro decanoyl-ACP thioesterase activity of 1.25 pmol 9min-~ 9 (embryo)-1 was obtained for C. lanceolata which is much less than the in-vivo incorporation of decanoic acid into seed triacylglycerols (83 pmol 9m i n - 1. (embryo)- ~; calculated from data of Bafor et al. 1990). A similar discrepancy can be calculated between the in-vitro lauroyl-ACP thioesterase activity (2.7 n m o i . m i n - 1. (cotyledon pair)- 1) and the in-vivo accumulation of lauric acid (56 n m o l - r a i n 1. (cotyledon pair)-1) in California bay seeds (calculated from data of Davies et al. 1991). These comparisons indicate that the in-vitro assay of M C T E in some way substantially underestimates the in-vivo activity. Possibly, the M C T E requires close association with the fattyacid synthase enzymes or other factors for optimal activity. In this regard, the purification of the Cuphea M C T E to homogeneity may not lead to results that have a better relation to in-vivo activities than the data obtained in this work. The Cuphea M C T E is most active for lauroyl- and myristoyl-ACP, and shows less activity for decanoyl-, palmitoyl-, stearoyl- and oleoyl-ACP. Presumably the binding site of the Cuphea M C T E has optimal affinity for acyl-ACPs with chain lengths of 10-14 carbon atoms whereas acyl-ACPs with 8 or less acyl carbon atoms are excluded. Decanoyl-ACP is therefore the first product of the acyl-ACP elongation by the fatty-acid synthase that will be hydrolyzed by the Cuphea MCTE. In Cuphea seeds the pools of 12:0-ACP, 14:0-ACP, 16:0-ACP and 18:0-ACp are very low (Singh et al. 1986), such that the 12:0-ACP and 14:0-ACP thioesterase activities of the M C T E cannot produce appreciable amounts of the respective free fatty acids. After elongation and desaturation to 18 : 1-ACP, the relatively high activity of the OTE may be essential to ensure that all 18:I-ACP will be hydrolyzed. It is puzzling that the chain-length specificity of the C. lanceolata and C. wrightii M C T E is almost identical, although the seed triacylglycerols of C. wrightii contain considerable amounts of lauric acid in additon to decanoic acid. Thus, these results emphasize the incomplete state of our understanding regarding the mechanistic details leading to the final fatty acid composition of seed storage triacylglycerols. The determination of fatty-acid chain length can be controlled by a variety of mechanisms. The condensing enzymes of most known soluble fatty-acid synthases show decreased rates of elongation when the acyl primer
431 reaches a length of either 16 or 18 carbons. In addition to condensing-enzyme specificity, in Saccharomyces cerevisiae, chain termination is accomplished when the length of acyl groups attached to the multifunctional fatty-acid synthase reaches 16 carbons and it is transferred from ACP to Coenzyme A by palmitoyl-transferase. In animals, termination o f fatty-acid synthesis is brought on by hydrolysis o f the acyl chain from the synthase by either a palmitoyl-specific thioesterase or in the case of mammary glands, by a medium-chain-specific enzyme. In plants, in addition to chain-length-specific acyl-ACP thioesterases, transfer o f fatty acids from ACP to glycerol-3-phosphate also serves as a termination reaction. In this study we have demonstrated that C. lanceolata and C. wrightii seeds contain at least two acylACP thioesterases, with one specific for oleoyl-ACP and one for medium-chain acyl-ACPs. In this regard, Cuphea is similar to California bay and, therefore, these results indicate that these two phylogenetically distinct species utilize a similar mechanism for the production of medium-chain fatty acids in seeds. This work was supported by grants of the German Federal Ministry for Research and Technology (F6rderkennzeichen 0319412 C), by the German Academic Exchange Service and by the National Science Foundation (grant number DCB 9005290). The authors would like to thank Dr. William Hitz, DuPont, for kindly providing anti-soybean OTE antibodies. Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.
References
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