Eur. J. Biochem. 210, 1015-1022 (1992)

0FEBS 1992

Short-chain and medium-chain aliphatic-ester synthesis in Sacckaromyces cerevisiae Philippe MALCORPS and Jean-Pierre DUFOUR Unite de Brasserie et des Industries Alimentaires, Universite Catholique de Louvain, Louvain-La-Neuvc, Bclgium (Received July 15iOctober 1, 1992) - EJB 92 1002

In the yeast Saccharnmyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 3 kDa and its apparent pZ is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-~~-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA : 0-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.

Short-chain and medium-chain aliphatic esters have been reported as by-products of oxidative metabolism in higher plants and fungi [I, 21. Although volatile aliphatic esters are important in producing the fruity aroma of foods and fermented beverages [3,4], the biochemical significancc of short-chain and medium-chain aliphatic-ester synthesis in cellular metabolism is still unknown. In most cases described in the literature, the synthesis of aliphatic esters involves alcoholysis of an acyl-CoA intermediate and is catalysed by acyltransferases or ester synthases. Such enzymes have been reported in various microorganisms [5 - 91 and during the ripening of banana [lo]. On the other hand, enzyme-catalysed esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A has been reported in human and rabbit myocardium [l I]. In the yeast Saccharomyces cerevisiae, esters are mainly synthesized and excreted under anaerobic fermentation conditions. It has been demonstrated that the synthesis of isoamyl acetate is catalysed by an acetyl-CoA : 0-isoamyl-alcohol acetyltransferase which is metabolically induced in the exponential phase of growth. The higher specific rate of ester production observed under anaerobiosis has been related to the induction of isoamyl-acetate synthase, the regulator of which is presumably linked to lipid metabolism [22]. It would be interesting to elucidate the molecular basis of ester synthase regulation, as ester synthesis could be one of the functions specifically induced during anaerobic growth of yeast [13]. Due to a lack of a rapid screening method for mutants affected in ester synthesis, characterization of ester synthases Correspondence to J.-P. Dufour, Unite de Brasserie et des Industries Alimentaires, UniversitP Catholique de Louvain, Place Croix du Sud 2jbte 7, B-1348 Louvain-La-Neuve, Belgium Fax: +3210472178. Abbreviation. BHT,butylated hydroxytoluene, 2,6-di-tert-butyl4-methylphenol.

is a prerequisite for the elucidation of their role in yeast metabolism. An ethyl-n-hexanoate synthase has been purified from Neurospora sp. [14]. Isoamyl-acetate synthase has been successfully s o h bilized from yeast membranous fractions, but recovery after purification was very low due to enzyme instability. Moreover, the final purified fraction was still crude [6, 91. In the present work, we present evidence that several enzymes are involved in the synthesis of short-chain and medium-chain aliphatic esters. We also describe the first purification to homogeneity and some physicochemical properties of a fully active isoamyl-acetate synthase from yeast.

MATERIALS AND METHODS Chemicals Sephacryl S-300, DEAE-Sephacel, Blue Sepharose CL6B, HiTrap Blue, PBE-94 and PB-74 were purchased from Pharmacia-LKB (Uppsala, Sweden). Bio-Gel HPT was from Bio-Rad (Richmond, CA, USA). The Thesit and n-dodecylmaltoside dctergents were from Boehringer Mannheim (Mannheim, FRG). N-Octyl-8-D-glucoside, acetyl-CoA and n-hexanoyl-CoA (lithium salts) were purchased from Sigma (St. Louis, MO, USA). 12-~~-Hydroxystearic acid was from Aldrich (Brussels, Belgium) and [l -14C]acetyl-CoA from Du Pont de Nemours, NEN Division (Dreieich, FRG). Culture conditions and cell-free extracts The yeast S. cerevisiae NCYC 366 was anaerobically grown on glucose at 28°C and harvested in the exponential phase of growth as previously described [12]. All subsequent operations were carried out on ice or at 4°C. Washed cells (75 g wet cells) were resuspended in 150 ml ice-cold homogenization buffer (0.25 M sucrose, SO mM Mes/Hepes, 10 mM

1026 EDTA, 5 mM dithiothreitol, pH 6.5) to which 1.5 ml 100 mM phcnylmethylsulfonyl fluoride (in dimethylsulfoxide) was added just beforc use. Aliquots of the resulting mixture (25 ml) were homogenized in a 75-1111 vial with a Braun MSK homogenizer as described elscwhere [15]. The decanted beads were washed twice with 5 ml buffer. The homogenates were pooled and centrifuged at 8000 x g for 15 min. The supernatant was further Centrifuged at 100000 x g for 1 h. Obtaining a crude protein precipitate from the cell-free extract

After diluting 250 ml of the supernatant (100000 x g , 1 h) fivefold in 1 1 of a solution containing 2.7 mM EDTA and 1 mM dithiothreitol at pK 6.5 (NaOH), ammonium sulfate (475 g) was slowly added under magnetic stirring. The pH was adjusted to 6.5 (NaOH) and the mixture centrifuged at 8000 x g for 20 iniii after 1 h of additional stirring. The pellets were pooled and diluted with 800 ml containing 125mM Na2HP04, 3.4mM EDTA. and 1.25mM dithiothreitol (pH 8.2, adjusted if necessary). The volume was brought to 1 1 with cold water and 250 ml poly(ethy1ene glycol) 6000 (50% masspol.) was added slowly under stirring. After 1 h of further stirring, the precipitate was collected by centrifugation at 8000 x g for 20 min. Partial resolution of ester synthase activities

Protein (20 mg) precipitated as described above was resuspended in 20 mM TrisiHCI. 3.6 M ethylene glycol, 50 mM NaCI, 1 mM EDTA and 1 mM dithiothreitol, pH 7.5 (buffer A). A solution of AT-dodecylmaltosidein buffer A was added to give final protein and detergent concentrations of 4 mg/ml and 9.8 mM, respectively. The suspension was centrifuged (10000 x g, 10 min) and 5 ml supernatant was loaded at a flow rate of 12 ml/h on a 1-ml-HiTrap Blue column equilibrated beforehand with buffer A containing 2 mM n-dodecylmaltoside. After washing the column with buffer A, unadsorbed proteins were pooled on the basis of the elution profile obtained by measuring the absorbance at 280 nm with a dual-path U V-2 monitor from Pharmacia-LKB. The column was further washed with 10 ml buffer A containing 3.4 mM Thesit instead of dodecylmaltoside, and isoamyl-acetate synthase was eluted with 0.6 M NaC1. Purification of isoamyl-acetate synthase

following buffer matrix: 3.6 M ethylene glycol, 3.4 mM Thesit. 6.9 pM BHT, 1 mM EDTA, 1 mM dithiothreitol. This buffer matrix was used for all solutions thereafter. Elution was carried out with 120 m M NaH2P04. Gel-permeation chromatography on Sephacryl S300 SF

Isoamyl-acetate-synthase-containing fractions (active fractions) eluted from the hydroxyapatite were pooled and layered onto a Sephacryl S300 SF column (2.6 cm x 78 cm) equilibrated with 20 mM TrisiHCl pH 7.5. Elution was carried out at 5.5 cm-I . h-'. Anion-exchange chromatography on DEAE Sephacel Active fractions from the gel-permeation chromatography were poolcd and loaded onto a DEAE-Sephacel column (1.6 cm x 3 6.5 cm) cquilibrated with the same buffer as above. After washing with 60 ml buffer, the adsorbed protcins were eluted with a 200-ml linear NaCl gradient (0- 150 mM) at 25 cm-' . h-I. Chromatography on Blue Sepharose CL-6B Active fractions from the anion-exchange chromatography were pooled and loaded onto a Blue Sepharose CL-6B column (1 cm x 9 cm) equilibrated with 30 mM Hepes/HCl, pH 7.5. After loading and washing with 30 ml of the same buffer, the column was eluted with a 60-ml linear NaCl gradient (0-1 M) at 9.9 cm-' . h-'. Affinity chromatography on Blue Sepharose CL-6B

Active fractions from the chromatography on Blue Sepharose CL-6B were concentrated from 25 ml to 4.5 ml on a small (1 cm x 2 cm) hydroxyapatite column, as described above, and diluted twice with a solution containing 3.6 M ethylene glycol, 3.4 mM Thesit, 6.9 pLM BHT, 1 mM dithiothreitol and 1 mM EDTA. This fraction was loaded onto a small Blue Sepharose column (1 cm x 2 cm) equilibrated with 30 mM NaH,P04 (pH 7.5). After washing with 10 ml buffer, the purified isoamyl-acetate synthase was clutcd with 8 ml buffer containing 0.5 mM dodecanoic acid and 0.1 mM acetyl-Co A. Isoelectric-point determination of isoumyl-ucetate synlhase

Solubilization with Thesit An aliquot of crude protein precipitate (from 75 g wet cells) was diluted to obtain a protein concentration of 10 mg/ ml in a solution containing 0.25 M sucrose, 50mM Mesi Hepes, 2 mM EDTA, 5 mM dithiothrcitol. 25.7 mM Thesit, 51 pM 2.6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene; BHT; 69m M stock solution in methanol) and 100 mM NaCl, pH 6.5. After 10 min ofmagnetic stirring, 0.25 vol. poly(cthy1ene glycol) 6000 (50% masspol.) was slowly added. After 1 h of additional stirring, the precipitate was discarded (1 1000 x g, 30 min).

A fraction obtaincd after solubilization and chromatography on hydroxyapatite was loaded onto the same Sephacryl300 SF column as described above, but equilibrated with a solution containing 3.6 M ethylene glycol, 1 mM EDTA, 1 mM NaN3, 3.4mM Thesit, 6.9 pM BHT, 1 mM dithiothreitol and 25 mM histidine/HCl (pH 6.4). Active fractions were loaded at 42 c1n-l . h - ' onto a PBE 94 column (1 cm x 36 cm), cquilibrated with the same buffer as above. Elution was carried out with 3.6 M ethylene glycol, 10% (by vol.) PB 74, 0.2 mM EDTA, 6.8 mM Thesit, 13.8 pM BHT and 2 m M dithiothreitol, pH 5.0 (HCl). Final recovery of isoamyl acetate synthase activity was 19%.

Chromatography on hydrox,ycrpatite

'

The supernatant was loaded (15 cm- h - ') on a Bio-Gel HPT hydroxyapatite column (1 cm x 8 cm) cquilibratcd with 30 mM HepeslHCl, pH 7.5. The column was washed with 20 ml containing 30 mM NaH2P04,pH 7.5 (NaOH), and the

Electrophoresis

SDS/PAGE was performed on 12.5% acrylamide slab gels [16]. Proteins were revealed by silver staining as described in ~71.

1017 Enzyme and protein assays Isoamyl-acetate synthase activity was measured by headspace gas chromatography as previously described [12, 181. 1 U activity corresponded to 1 pmol isoamyl acetate synthesizedlh. Ethyl-acetate synthase and ethyl-n-hexanoate synthase assays were carried out at 30°C in a medium (200 pl) containing 200mM KH,PO, (pH 7.8), 2 m M EDTA, 0.513 M ethanol and 0.3 mM acetyl-CoA or 50 pM n-hexanoyl-CoA. Under all conditions, the enzyme activities werc proportional to the amount of protein added and to the incubation time. Rcactions were stopped by lowering the pH to 3.0 with 5.5 pI 3 M H2S04. Protein content was determined according to Bradford [19]. In the presence of Thesit, proteins were precipitated [20] and solubilized in 5 % SDS (3 min, l00OC) before determination according to the bicinchoninic acid method [21]. After SDSiPAGE, the protein content of the purified isoamyl-acetate synthase preparation was estimated with a PhamaciaLKB 2222-020 Ultroscan Densitometer by comparing silverstained band intensities with protein standards. Enzymic acetylation of 12-~~-hydroxystearic acid 'lhe reaction was carried out in a final volume of I00 pl containing 100 mM KH2P04, 30 mM n-octylglucoside, 0.5 mM 12-~~-hydroxystearic acid (50 mM stock solution in dimethylsulfoxide), 80 pM [l-14C]acetyl-CoA (50.6 Ci/mol) and 5 - 20 pl purified isoamyl-acetate synthase preparation (2.8 Limo1 isoamyl acetate . h - ' . m1-I). After 30 min of incubation at 30 "C, the reaction was stopped by lowering the pH to 3.0 by addition of 2.5 p1 3 M H2S04. The reaction product was extracted three times with 100 p1 diethyl ether, and the organic phase was washed with 100 p1 water. Ether was evaporated under nitrogen and the residue resuspended in chloroform. Radioactivity was measured by scintillation counting in Betafluor (National Diagnostics, Highland Park, NJ) scintillation liquid. The purity of the 12-[1-'4C]acetoxystearic acid synthesized was checked by thin-layer chromatography on silica plates developed with hexane/acetic acid (96 : 4 by vol.) and autoradiographed overnight on XOMAT film (Eastman Kodak Co., Rochester, NY). 1 2 - ~ Acetoxystearic acid was obtained by acetylation of 12-DLhydroxystearic acid with acetic anhydride in pyridine.

Table 1. Comparison between in vivn and in v i m specific activities. The in vivo activitics were measurcd as dcscribcd in [I21 in the presence of 0.5 Methanol or 1 mM isoamyl alcohol. Theisoamyl-acetate synthase activity (in vitro) was measured as described under Materials and Methods.

Sample

Ester synthase activity ethyl acetate

isoamyl acetate

ethyl-nhexanoate

nmol . h-' . mg protein-'

In vivo I n vitro a

45 36

0.6 1.5

6.5 5"; 237

1 mM isoamyl alcohol.

15 mM isoamyl alcohol

exponential phase of anaerobic growth. In vitro specific synthesis rates of isoamyl acetate, cthyl n-hexanoate and ethyl acetate were coinpared with data obtained in viva as previously described [12]. When measured in the presence of alcohol concentrations similar to those found in fermentation (1 mM isoamyl alcohol and 0.5 M ethanol), the values measured in vitro were rather low but of the same order of magnitude as that observed in vivo (Table 1). We conclude that the in vitro assays of ester synthase activity were meaningful. To improve the detcction limits of thc assay during purification of isoamyl acetate synthase, the assay was routinely performed in the presence of 15 mM rather than 1 mM isoamyl alcohol. The appropriateness of these assay conditions has been demonstrated [ 121. 'The homogenate was fractionated by high-speed centrifugation (Table 2). Almost 50% of the ester synthase activities remained in the 'soluble' fraction (100000 x g , 1 h supernatant). This fraction was precipitated with ammonium sulfate and poly(ethy1ene glycol) 6000 to further characterize the activities present in the fraction. Ester synthase activities precipitated in a fraction containing high-molecular-mass proteins and lipoproteins. This is in keeping with preliminary investigations of the precipitate by gel-permeation on a Sephacryl S300HR column (fraction~ chromatography ation range; 10 - 1500 kDa), in which the ester synthase activities eluted in the void volume of the column (data not shown). Partial resolution of the ester synthase activities

RESULTS Ester synthase activities in Saccharomyces cevevisiae Ester synthase activities were measured in a crude cellfrec extract obtained froin cells harvested at the end of the

The isoamyl-acetate synthase activity recovered in the poly(ethy1ene glycol) 6000 precipitate can be completely solubilised with n-octylglucoside, as demonstrated earlier [22]. Unfortunately, the ethyl-n-hexanoate synthase activity was

Table 2. Distribution of ester synthase activities after centrifugation of a 5'. cerevisiue homogenate for 1 h at 100000 xg. The activities were measured with 5-pI (ethyl acetate) or IO-pI (isoamyl acetate and ethyl n-hexanoate) for 10 min (ethyl acetate), 15 min (isoamyl acctatc) or 25 min (ethyl n-hexanoate). Other conditions are described under Materials and Methods. Sample

Volume

Protein

Ester synthase activity

ethyl acetate

isoamyl acctate

ethyl n-hexanoate

21 1

1405 660 563

8.9 4.5 1.5

ml

Hoinogenate Supernatant Pellet

210 243

I0

5928 3616 1956

143 81

1018 Table 3. Resolution of the ester synthase activities by affinity chromatography. The activities were measured with 50-pl (ethyl acetate and ethyl n-hexanoate) or 10-pI (isoamyl acetate) and for 30 min (ethyl acctatc and cthyl n-hexanoate) or 15 min (isoamyl acetate). Samplc

Volume

Bn7yme activity with

ethyl n-hexanoate

Crude protein precipitate Solubilized fraction HiTrap Blue chromatography unadsorbed proteins eluted proteins (0.6 M NaCI)

ethyl acetate

isoamyl acetate

ml

pmolih

4.2 5

25.1 39.5

1.42 0.76

10.7 9.3”

7.8 7.7

29.3 5.9

0.48 0.76

0.53” 13.1

Reaction medium contained 0.8 mM Thesit.

unstable under these conditions and remained insoluble (data not shown). Unlike n-octylglucoside, n-dodecylmaltoside allowed to stabilizc the ethyl-n-hexanoate synthase activity and to solubilize the isoamyl-acetate synthase. Under these conditions, the solubilized isoamyl-acetate synthase could be adsorbed on a HiTrap Blue column but required 2 M NaCl to be eluted. Inhibition by n-dodecylmaltoside and high-salt inactivation were responsible for low recovery of this activity (data not shown). However, the isoamyl-acetate synthase activity could be eluted with 0.6 M NaCl and fully recovered when n-dodecylmaltoside was replaced by Thesit in the elution buffer. When the precipitate was solubilized by ndodecylmaltoside and fractionated by affinity chromatography on a HiTrap Blue column following this procedure (Table 3), most of the ethyl-n-hexanoate synthase activity was recovered in the unadsorbed protein fraction which lacked any significant isoamyl-acetate synthasc activity. A small part of the ethyl-n-hexanoate synthase activity was also eluted from the column with 0.6 M NaCl. Ethyl-acetate synthase activity was found in both fractions (unadsorbed and adsorbed). We conclude that isoamyl acetate and cthyl n-hexanoate are synthesized by distinct enzymes that are differently affected by detergent. Thereafter, our main focus was on purifying the isoamyl-acetate synthasc, to determine whether this enzyme could synthesize ethyl acetate and small amounts of ethyl nhexanoate or whether another enzyme was involved.

in ,

I

K

vo

I

0

50

250

(ml)

5

0

Fraction 0

a,-

-E E-

10

number

15

0.10

20

(5.6mlifraction)

10

l

8

“I;

m f

6

0

N m

m o

Z E

Isoamyl-acetate synthase was purified from the ‘soluble’ proteins precipitated as described under ‘Materials and Methods’ and was selectively solubilizcd from lipoproteins with Thesit. During the solubilization step, the bulk of the contaminants remained associated as high-molecular-mass aggregates which were removed with the pellet after a second poly(ethy1ene glycol) 6000 precipitation (data not shown). The solubilized isoamyl-acetate synthase was then concentrated by adsorption to hydroxyapatite, which increased the purification factor. Fig. 1 shows typical elution profiles for gelpcrmeation chromatography on Sephacryl S300 SF (Fig. 1A), the anion-exchange chromatography on DEAE-Sephacel (Fig. 1B) and chromatography on Blue Sepharose CL-6B (1 C). The overall purification scheme used for isoamyl-acetate synthase is summarized in Table 4. The specific activity of the enzyme rose from 0.38 pmol . h - l . mg protein-’ in the cellfree extract to 6350 pmol . h-’ . mg protein-’ in the purified fraction. This amounts to a 19000-fold purification. Three

300

,

20 I

m

C

200

150

100

Volume

I

Purification and characterization of isoamyl-acetate synthase

020

$2

Q 4

x=”

5?

0 -

2 : 0

5

Fraction

10

number

15

20

25

(1.8ml/fractlon)

Fig. 1. Purification of isoamyl-acetate synthase from S.cerevisiae. (A) Gel-permeation chromatography on Sephacryl S300 SF; (B) anionexchange chromatography on DEAE-Sephacel; (C) chromatography on Blue Sepharose CL-6B. The bar indicates the active rractions. For details see the text. (0)Absorbance at 280 nm; ( 0 )isoamyl-acetate synthase activity.

purifications have been carried out with satisfactory repeatability. The same electrophoretic pattern and final activity recovery of 4- 10% were obtained. The high specific activity increase obtained suggests that the enzyme is present in very small amount in the yeast cell.

1019 Table 4. Purification of isoamyl-acetate synthase from S. ceuevisiue. PEG, poly(ethy1ene glycol) Sample

Cell-free extract 100000 x g , 1 h supernatant Precipitated fraction (PEG) Solubilized fraction (Thesit) Hydroxyapatite Gel permeation DEAE-Sephacel Blue Sepharose Affinity on Blue Sepharose

Volume

ml 280 260 54.5 142 8 46 48 25 5.4

Proteins

mg 7168 5304 1199 35.5 18.2 10.4 1.06 0.05 0.02

Isoamyl-acetate synthase activity total

yield

pmol/h 2425 1456 946 469 386 429 432 280 127

%

100 60 39 19.3 15.9 17.7 17.8 11.5 5.2

Specific activity

Purification factor

Fmol. h- ' . mg-' 0.38 0.31 0.79 13.2 21.2 41.3 408 5600 6350

-fold

1 0.8 2.1 35 56 109 1074 14700 19200

6.5

6.0

Y

r

a

5.5

5.0

0

10

Fraction

20 number

30

40

(5.2mllfraction)

Fig. 3. Elution profile of isoamyl-acetate synthase after chromatofocusing on PBE 94. Conditions were as described under Matcrials and Methods. (0)pH at 4°C; ( 0 )activity.

Stability

Fig. 2. SDS/PAGE of isoamyl-acetate synthase. Before electrophorcsis, samples werc boiled for 3 min in loading buffer containing 1% (massjvol.) dithiothreitol. (a) Partially purified enzyme aftcr chromatography on hydroxyapatite (0.1 5 pmol/h). (b) Molecular mass standards: (from the top) phosphorylasc b (94 kDa); albumin (67 kDa); ovalbumin (43 kDa); carbonic anhydrase (30 kDa); trypsin inhibitor (20.1 kDa). (c) Purified enzyme after affnily chromatography on Blue Sepharose CL-6B (1.5 pmol/h).

Properties of isoamyl-acetate synthase

Molecular mass and p l The electrophoretic patterns of the enzyme preparations from the first hydroxyapatite chromatography and from the last affinity chromatography on Blue Sepharose CL-6B, are shown in Fig. 2. Isoamyl-acetate synthase was purified to apparent homogeneity and was found to consist of a single peptide with a molecular mass of 57 f 3 kDa (Fig. 2). The apparent p l of the native enzyme was 5.5 (Fig. 3). Relative to p l determination, one has to point out that under the acidic conditions used, the enzyme was unstable and underwent aggregation (data not shown).

The enzyme exhibited an absolute requirement for ethylene glycol or glycerol in order to maintain its activity after a freeze/thaw cycle. Long-term stability was improved by including Thesit (3.4 mM), EDTA (1 mM) and dithiothreitol (1 mM), and by maintaining the pH above 7.0. Under these conditions, no significant loss of activity was detected after 24 h on ice or after six months at - 30 "C. Eflects of substrate concentration The purified isoamyl-acetate synthase was able to catalyse the synthesis of ethyl acetate when ethanol was used as cosubstrate. Attempts to detect ethyl n-hexanoate synthesis with ethanol and n-hexanoyl-CoA werc unsuccessful, even in the presence of n-dodecylmaltoside. Typical MichaelisMenten saturation curves were obtained for both activities with increasing concentrations of acetyl-CoA (Fig. 4). EadieHofstee plots of the corresponding data (insets of Fig. 4A and B) were used to calculate K,,,(app) values for acetyl-CoA: 25 pM and 45 pM for the synthesis of isoamyl acetate and ethyl acetate, respectively. Isoamyl acetate synthesis increased with the isoamyl alcohol concentration and gave an apparent saturation curve with a K,(app) of 25 mM for isoamyl alcohol. Ethyl acetate synthesis increased with ethanol concentration up to 1 M and decreased thereafter (Fig. 5).

1020

2 7 100 a

f L

=. E 80

I: ' '"''\,j

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ac

I-

:: z-

60

40

40

-40

-r Z

I

z -

8

20

20

0

1

2 VI s

3

4

20

0

0

100

50

150

250

200

300

12

0

100

200

300

500

400

600

I

B

l l VIS

0 100

0

200

500

400

300

600

0

100

200

300 400 5 0 0

600

200

400

700

800

900

(pM)

Acetyl-CoA

Fig. 4. Enzyme activity versus acetyl-CoA concentration plots for the synthesis of isoamyl acetate (A) and ethyl acetate (B) with purified isoamyl-acetate synthase. Insets show the corresponding EadieHofstee plots. Reaction media contained 15 mM isoamyl alcohol (A) or 0.51 M ethanol (B). Incubation was carried out for 15 min (A) or 10 min (B) with 5 p1 diluted, purified enzyme preparation. The purificd enzyme, with an activity of 100 pmol isoamyl acetate h-' ml-' (assay conditions as described under Materials and Methods), was diluted 20-fold (A) or fourfold (B) in the last chromatography buffer without acetyl-CoA or dodccanoic acid (see Materials and Methods). ( 0 )Isoamyl-acetate and ( A ) ethyl-acetate synthase activities.

0

100

300

n-Hexanoyl-CoA

500

600

(pM)

Fig. 6. Enzyme activity versus n-hexanoyl-CoA concentration plots for the synthesis of ethyl n-hexanoate with the crudc proteins precipitated by ammonium sulfate (A), the unadsorbcd (B) and the eluted (C) protein fractions after chromatography on a HiTrap Blue column. Assays were carried out (A) for 15 min with 10 p1 sample (300 pg proteins) or (R) for 30 min with 50 p1 sample.

12-Hydroxystearic acid acetylation

0

u 0.0

0 l

0

1 Ethanol

-

r

10

.

,

20

,

3

2

(M) ,

30

lsoamyl alcohol

.

,

40

,

,

50

(mM)

Fig. 5. Enzyme activity versus isoamyl alcohol or ethanol concentration plots for the synthesis of isoamyl acetate and ethyl acetate with purified isoamyl-acetate synthase. Keaction media contained 300 pM acetylCoA. Isoamyl acetate synthesis was carried out by incubating 5 pl purified enzyme preparation (5 pmol isoamyl acetate . h-' . ml-' using assay conditions a described under Materials and Methods), for 15 min. Ethyl acetate synthesis was carried out by incubating 45 pl purificd enzyme preparation for 20 min. ( 0 )Isoamyl-acetate and ( A ) ethyl-acetate synthase activities.

Thc purified isoamyl-acetate synthase acetylated the hydroxy moiety of 12-hydroxystearic acid. The activity of the purified isoamyl-acetate synthase preparation in the presence of 0.5 mM isoamyl alcohol or 0.5 mM 12-hydroxystearic acid was 0.15 pmol . h-' . ml-' or 0.20 pmol h-' . ml- ', respectively. The isomeric form of the product was not determined. Synthesis of 12-acetoxystearic acid was proportional to the isoamyl-acetate synthase activity added (not shown). Kinetic data for other ethyl-ester synthases

The ethyl-n-hexanoatc synthasc activity curvc for the crude protein precipitate demonstrated a bimodal kinetic pattern when the n-hexanoyl-CoA concentration was varied. Under these conditions, two K,,,(app) were determined: 15 pM and 80 pM (Fig. 6A). After solubilization of the protein pre-

1021

Ethanol

(M)

Pig. 7. Enzyme activity versus ethanol concentration plots for the synthesis of ethyl acetate and ethyl a-hexanoate with the unadsorhcdprotcin fraction after chromatography on HiTrap Blue. Reaction mcdia contained 300 pM acetyl-CoA or 170 pM n-hexanoyl-CoA. Incubation was carried out for 30 min with 50 p1 sample. ( A ) Ethyl-acetate and (0) ethyl-n-hexanoate synthase activities.

(u

m

5 z

0 1 0

r

I

10 15 20 25 Preincubation lime at 45°C (mln )

5

I 30

Fig. 8. Heat-inactivation of the ethyl-acetate and ethyl-n-hexanoate synthase activities of the unadsorbed protein fraction after chromatography on HiTrap Bluc. Samples were incubated a t 45 "C for the indicated time then the residual activities measured with SO pl sample for 30 min. Other conditions were as described under Materials and Methods. (0) Ethyl-n-hexanoate and ( A ) ethyl-acetate synthasc activi tics.

cipitate with n-dodecylmaltoside, fractionation of the extract on HiTrap Blue made it possible to separate two peaks of ethyl-n-hexanoate synthase activity: one in the unadsorbed protein fraction and one desorbed with 0.6 M NaCl (Table 3 ) . The corresponding K,,,(app) values for n-hexanoyl-CoA were 18 pM (unadsorbcd fraction) and 150 pM (desorbed fraction) (Fig. 613 and C). The unadsorbed protein fraction was unable to catalyse the synthesis of isoamyl acetate but exhibited ethylacetate synthasc activity with a K,(app) of 6 pM for acetylCoA (not shown). Moreover, ethyl n-hexanoate synthesis increased in this fraction up to an ethanol concentration of 2 M, decreasing thercaftcr (Fig. 7). Unlike ethyl n-hexanoate synthesis, ethyl acetate synthesis increased up to 2.7 M ethanol and no saturation or substrate-inhibition pattern was observed in the range of concentrations tested. Lastly, the two cstcr synthase activities exhibited different heat-inactivation profiles (Fig. 8). DISCUSSION Despite the high sensitivity of thc gas/liquid chromatography method used in this work, the rates of ester synthesis

were difficult to measure because of the low specific activities in cell-free extracts. Detection of ethyl-ester synthase activity was improved by use of high substrate (ethanol) concentrations (up to 1 M), which in fact correspond with the values commonly observed during alcohol fermentation. One should bear in mind that, as observed with estcrascs or lipases, the resulting ester synthesis could be due to non-physiological deacylation of acyl-enzyme intermediates by alcohols [24]. The specific activities of the ethyl-ester synthases measured here are in the same range of magnitude as in vivo activities and relatively high affinities were determined for the acetylCoA or acyl-CoA co-substrate. These observations suggest that we did measure specific enzyme-catalysed reactions. So far, the molecular approach to understanding the biochemical significance and regulation of aliphatic-ester synthesis has been restricted by the lack of a rapid screening method for mutants modified in ester synthesis. Therefore, identification and purification of an ester synthase is the starting point for cloning the structural gene of such an enzyme. The properties of the isoamyl-acetate synthase purified here to homogeneity, resemble those of crude preparations obtained by others [6, 91. The enzyme can be defined as an acetyl-CoA :0-alcohol acetyltransferase because of its inability to use acyl-CoA as co-substrate. The activity distributed between the pellet and the supernatant after high-speed centrifugation. Previous studies have shown this enzyme to be associated with a slowly sedimenting or floating structure of lipoprotein nature released during vigorous disruption of cells with glass beads [lS, 221. The partially purified enzyme from the pellet could not be distinguished from the purified enzyme isolated from the soluble fraction used in this work, when comparing apparent native molecular mass by gel-permeation chromatography, p l and kinetic properties (data not shown). Two enzymes were found to catalyse ethyl n-hexanoate synthesis in the lipoprotein fraction. The high sensitivity of their activity towards detergents suggests a strongly hydrophobic nature. Their behaviour thus differs from that of the soluble ethyl-n-hexanoate synthase rec.ently described in Ncurospora sp. [14]. In S . cerevisiae, we have observed that the synthesis of ethyl acetate is catalysed by isoamyl-acetate synthase and by a second enzyme present in a protein fraction devoid of significant isoamyl-acetate synthasc activity and containing most of the ethyl-n-hexanoate synthase activity. However, the ethyl-n-hexanoate and ethyl-acetate synthase activities differ as to the effect of ethanol concentration on their reaction rates and to their heat-denaturation profiles. This suggests the existence of two distinct enzymes. Unlike ethyl-n-hexanoate synthase, ethyl-acetate synthase exhibited no saturation by the substrate ethanol and was not inhibited at high substrate concentrations. Such linear kinetic patterns have been described by others [7, l l ] and are comparable to the dependence of hydrolases on the substrate water. For ethyl-n-hexanoate and isoamyl-acctatc synthases, we have not yet investigated the distinction between true inhibition of the enzyme by excess of substrate or inactivation by denaturation. A possible role of ethyl acetate synthesis could be to regenerate free coenzyme A from acetyl-CoA without releasing acetic acid. The physiological role of medium-chain aliphaticester synthesis (isoamyl acetate, ethyl n-hexanoate) in yeast remains undetermined. Ethyl n-hexanoate could be a by-product of the synthesis of long-chain saturated-fatty-acid ethyl esters, which might be specific to strictly anaerobic growth and stored like the triglycerides of cells grown aerobically [25]. Interestingly, acetylation of long-chain alcohols has been

1022 reported in the storage or subcellular targcting of dolichols [26] and in the formation of intermediates in pheromone biosynthesis [27]. As demonstrated in this work, several distinct enzymes could be involved in ester synthesis in yeast. This suggests possibly different metabolic roles for long-chain and mediumchain alcohol acetylation and for esterification of long-chain and medium-chain fatty acids with ethanol and ethyl acetate synthesis. In S. cerevisiae, aliphatic-ester synthesis is associated with anaerobic growth. An interesting property of isoamyl-acetate synthase is ils ability to acetylate 12-~~-hydroxystearic acid. This fatty acid has been reported to be acetylated and to support yeast growth under anaerobic fermentation conditions, as do unsaturated fatty acids [28]. Acetylation of the hydroxy moiety could be a prerequisite to incorporation of hydroxy fatty acids into cell-membrane phospholipids. If so, it should be possible to select conditional mutants defective in the acetylation reaction and which have lost isoamyl-acetate synthase activity. Previous work has demonstrated simultaneous repression of isoamyl-acetate, ethyl-acetate and ethyl-n-hexanoate syntheses by trace amounts of oxygen and/or unsaturated fatty acids [I 21. The involvement ofdistinct enzymes reinforces our hypothesis that a common regulatory mechanism is involved which probably acts at gene level. Unsaturated fatty acids could be considered effectors of yeast anaerobic metabolism, like haem which mediates regulation of gene expression by oxygen in S . cevevisiae under aerobiosis [I 31. To our knowledge, this type of interaction has never been described before in the anaerobic metabolism of yeast.

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Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly re...
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