Functional characterization of enone oxidoreductases from strawberry and tomato fruit

Article abstract of DOI:10.1021/jf071055o

Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase, is a ripening-induced, negatively auxin-regulated enzyme that catalyzes the formation of 4-hydroxy-2,5-dimethyl- 3(2H)-furanone (HDMF), the key flavor compound in strawberry fruit by the reduction of the α,β-unsaturated bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF). Here we show that recombinant FaEO does not reduce the double bond of straight-chain 2-alkenals or 2-alkenones but rather hydrogenates previously unknown HMMF derivatives substituted at the methylene functional group. The furanones were prepared from 4-hydroxy-5-methyl-3(2H)-furanone with a number of aldehydes and a ketone. The kinetic data for the newly synthesized aroma-active substrates and products are similar to the values obtained for an enone oxidoreductase from Arabidopsis thaliana catalyzing the α,β-hydrogenation of 2-alkenals. HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in tomato and pineapple fruit by HPLC-ESI-MSⁿ and became ¹³C-labeled when D-[6-¹³C]-glucose was applied to the fruits, which suggested that a similar HDMF biosynthetic pathway occurs in the different plant species. With a database search (http://ted.bti.cornell.edu/ and http://genet.imb.uq.edu.au/Pineapple/), we identified a tomato and pineapple expressed sequence tag that shows significant homology to FaEO. Solanum lycopersicon EO (SIEO) was cloned from cDNA, and the protein was expressed in Escherichia coli and purified. Biochemical studies confirmed the involvement of SIEO in the biosynthesis of HDMF in tomato fruit.

Full text of DOI:10.1021/jf071055o

J. Agric. Food Chem. 2007, 55, 6705−6711 6705
Functional Characterization of Enone Oxidoreductases from
Strawberry and Tomato Fruit
DOROTHEÄ E KLEIN,† BARBARA FINK,† BEATE AROLD,†
WOLFGANG EISENREICH,‡ AND WILFRIED SCHWAB*,†
Biomolecular Food Technology, TU Mu¨nchen, Lise-Meitner-Strasse 34, 85354 Freising, Germany, and
Organic Chemistry and Biochemistry, TU Mu¨nchen, Lichtenbergstrasse 4,
D-85748 Garching, Germany
Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxi-
doreductase, is a ripening-induced, negatively auxin-regulated enzyme that catalyzes the formation
of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), the key flavor compound in strawberry fruit by
the reduction of the R,â-unsaturated bond of the highly reactive precursor 4-hydroxy-5-methyl-2-
methylene-3(2H)-furanone (HMMF). Here we show that recombinant FaEO does not reduce the double
bond of straight-chain 2-alkenals or 2-alkenones but rather hydrogenates previously unknown HMMF
derivatives substituted at the methylene functional group. The furanones were prepared from
4-hydroxy-5-methyl-3(2H)-furanone with a number of aldehydes and a ketone. The kinetic data for
the newly synthesized aroma-active substrates and products are similar to the values obtained for
an enone oxidoreductase from Arabidopsis thaliana catalyzing the R,â-hydrogenation of 2-alkenals.
HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in
tomato and pineapple fruit by HPLC-ESI-MSn and became 13C-labeled when D-[6-13C]-glucose was
applied to the fruits, which suggested that a similar HDMF biosynthetic pathway occurs in the different
plant species. With a database search (http://ted.bti.cornell.edu/ and http://genet.imb.uq.edu.au/
Pineapple/), we identified a tomato and pineapple expressed sequence tag that shows significant
homology to FaEO. Solanum lycopersicon EO (SlEO) was cloned from cDNA, and the protein was
expressed in Escherichia coli and purified. Biochemical studies confirmed the involvement of SlEO
in the biosynthesis of HDMF in tomato fruit.
KEYWORDS: Fragaria x ananassa; Solanum lycopersicon; enone oxidoreductase; 4-hydroxy-2,5-dimethyl-
3(2H)-furanone; flavor formation
INTRODUCTION
5-methyl-3(2H)-furanone (HMF, norfuraneol), and 5-(or 2)-
ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone (EHMF, homo-
furaneol) all contain this structural element capable of forming
a strong hydrogen bond and rank among the important flavor
chemicals (Figure 1). These compounds are known to be
products of the Maillard reaction between reducing sugars and
amino acids and are common to a variety of thermally processed
foods, but they have also been identified as natural constituents
of fruits, insects, and fermented products, implying a biochemi-
cal formation pathway (1).
The first indication for the enzymatic formation of HDMF
in strawberry fruit was provided by a study demonstrating the
correlation between fruit-ripening stage and HDMF concentra-
tion (7). Incorporation experiments with radiolabeled precursors
and substances labeled with stable isotopes revealed D-fructose-
1,6-diphosphate as an efficient biogenetic precursor of HDMF
(4, 8). In strawberry, D-fructose-1,6-diphosphate is converted
by an as yet unknown enzyme to 4-hydroxy-5-methyl-2-
methylene-3(2H)-furanone (HMMF), which serves as substrate
for an oxidoreductase recently isolated from ripe fruit (9).
Among the volatiles that constitute strawberry (Fragaria x
ananassa) flavor, 4-hydroxy-2,5-dimethyl-3(2H)-furanone
(HDMF, Furaneol) is the most important because of its high
concentration (up to 55 mg/kg fruit fresh weight) and low odor
threshold (10 ppb) (Figure 1) (1). HDMF has a distinctive sweet
caramel-like flavor and was first isolated from pineapple
(Ananas comosus) (2) and then from strawberry (3) and a
number of different fruits (1). Radiotracer studies demonstrated
the bioconversion of HDMF to its methyl ether, its â-D-
glucopyranoside, and subsequently to the malonylated derivative
of HDMF glucoside (4, 5).
It has been postulated that compounds eliciting a caramel-
like aroma have a planar enol-carbonyl substructure in a cyclic
dicarbonyl compound (6). The heterocycles HDMF, 4-hydroxy-
* Corresponding author. Telephone: +49(0)8161548-312. Fax: +49-
(0)8161548-595. E-mail: schwab@wzw.tum.de.
† Biomolecular Food Technology.
‡ Organic Chemistry and Biochemistry.
10.1021/jf071055o CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/18/2007

6706 J. Agric. Food Chem., Vol. 55, No. 16, 2007
Klein et al.
Figure 1. Chemical synthesis of substrates and kinetics of FaEO and SlEO. HMMF, EDHMF, HMPDF, BDHMF, and HMMEF starting from HMF and
structures of the products HDMF, EHMF, HMPF, BHMF, and HIMF. Enzymatic assays were carried out with final concentrations of unsaturated
hydroxyfuranones in the range of 0.14
7. The assays were run for exactly 35 min at 30
calculated as “EHMF equivalents”. Km and Vmax values were determined by fitting the data to the Hanes equation. All data points are averages of
duplicates. nd not determined, na not applicable.
−
5.7 mM in mixtures containing 1.5
µg of FaEO (or SlEO) and 12 mM NADH in 250 µL of phosphate buffer pH
°
C, and HPLC-ESI-MSn was performed immediately. The amounts of formed HMPF and BHMF were
)
)
JM109 Escherichia coli cells. The vector was purified and digested
with endonucleases XhoI and NcoI, and after cutting off the phosphate
groups, the insert was ligated into an XhoI and NcoI predigested
expression vector pET29a(+) (Novagen, Calbiochem-Novabiochem
GmbH, Schwalbach, Germany). The resulting recombinant plasmid was
amplified in E. coli JM109 cells. Following sequencing they were
transformed into BL21(DE3)plys E. coli host strain for expression.
Expression and Purification. The cloning and isolation of the full-
length strawberry cDNA FaEO was conducted as described by Raab
et al. (9). The expression and purification of the enzymes FaEO and
SlEO were performed according to a published protocol (9).
Sequence analysis of two peptide fragments showed identity
with the protein sequence of a strongly ripening-induced, auxin-
dependent putative quinone oxidoreductase (FaQR). Because
the recombinant protein reduced only the artificial substrate 9,-
10-phenanthrene quinone (Km ) 35 µM) out of a number of
1,2- and 1,4-quinones tested and formed HDMF from HMMF,
the enzyme was renamed enone oxidoreductase (FaEO) (9).
In the present study, we report the synthesis and characteriza-
tion of HMMF, the natural substrate of FaEO, and its derivatives
as well as their biochemical conversion by FaEO. The identi-
fication of HMMF in HDMF-forming fruits led to the cloning,
heterolog expression, and characterization of an enone oxi-
doreductase from Solanum lycopersicon (SlEO), which catalyzes
the same reactions as FaEO.
Synthesis and Purification of HMMF and Its Derivatives. HMF
(16.9 g, 43.8 mmol), copper(II)acetate (20 mmol), sodium acetate (25
mmol), and 12 mL of an aqueous formaldehyde solution (37%) were
dissolved in 75 mL of acetic acid. For the synthesis of the derivatives,
the formaldehyde solution was replaced by acetaldehyde (6.5 g, 148
mmol), propionaldehyde (7.1 g, 148 mmol), butyraldehyde (10.6 g,
148 mmol), or acetone (7.1 g, 148 mmol). The solution was heated to
50 °C and stirred for 1 h before being diluted with 150 mL of water.
Products were isolated by diethyl ether (100 mL) extraction. The extract
was dried over sodium sulfate and concentrated by distillation. The
residue was again dissolved in 50 mL of water, extracted with 50 mL
of diethyl ether, and concentrated to remove remaining acetic acid.
Purification was performed by preparative silica gel thin layer chro-
matography (silica gel 60 F 254, 5 × 20 cm, 0.25 mm, Merck,
Darmstadt, Germany) with pentane/diethyl ether 1:1 (v/v). The bands
of interest were removed and extracted with diethyl ether. Products
were finally purified by preparative RP18-HPLC-UV, extracted with
diethyl ether, and analyzed by NMR.
Synthesis and Purification of the HMMF Thioether Adduct.
HMMF (ca. 0.4 mmol) and 3-mercaptobenzoic acid (MBA) (0.39
mmol) were added to 10 mL of water, and the solution was stirred in
the dark at room temperature overnight. The progress of the reaction
was monitored by HPLC-ESI-MSn, and the product was purified by
preparative RP18 chromatography (16 × 4 cm, LiChrospher 100) using
solvent mixtures consisting of water and acetonitrile (ACN) of
decreasing polarity. The HMMF-MBA product eluted with 50% ACN.
The fractions were combined and extracted with 50 mL of diethyl ether.
The extract was dried over sodium sulfate, concentrated, and subjected
to silica gel chromatography (25 × 2.5 cm, silica gel 60, 0.04-0.063
MATERIALS AND METHODS
Materials. All reagents and solvents were obtained from Sigma-
Aldrich, Taufkirchen, Germany, unless otherwise stated. D-[6-13C]-
glucose was obtained by Deutero GmbH, Kastellaun, Germany.
Plant Material. Strawberry fruits (Fragaria x ananassa Elsanta)
were harvested at three different developmental stages (full sized green
fruits, turning stage fruits, and full-ripe red fruits) from a commercial
field. Cherry tomato (Solanum lycopersicon cerasiforme) plant material
was harvested in different stages (buds, flowers, and fruits) from a
private garden or was bought from a local market. Pineapple fruits
(Ananas comosus) were obtained from a local supermarket.
RNA Isolation. Isolation of total RNA from different tissues of
cherry tomatoes was performed with the RNeasy Kit (Qiagen, Hilden,
Germany) following the manufacturer’s instructions.
cDNA Synthesis. The SlEO cDNA was synthesized using Super-
Script III (Invitrogen, Karlsruhe, Germany) for RT-PCR and the specific
primers 5′-GAC CAT GGG TAT GGA AGC TCT GTT ATC TTC-3′
containing the restriction site NcoI and 5′-GAC TCG AGA GGA ATG
GGA TGA ATA ACA AC-3′ containing XhoI. PCR products were
separated on a low melting agarose gel (1.5%). Bands of about 1200
bp were cut from the gel and purified by QIAquick Gel Extraction Kit
(Qiagen). After ligation of the A-tailed insert into pGem T easy
(Promega, Mannheim, Germany), the plasmid was transformed into

Enone Oxidoreductases
J. Agric. Food Chem., Vol. 55, No. 16, 2007 6707
mm, Carl Roth GmbH, Karlsruhe, Germany) using mixtures of pentane
and diethyl ether of increasing polarity. The product eluted with 100
mL of diethyl ether. The fractions were combined, concentrated, and
analyzed by NMR.
0.25 µm). The GC parameters were as follows: Initial temperature of
40 °C for 5 min, increased to 250 °C at 5 °C/min intervals. The helium
gas flow rate was 3 mL/min. The EI-MS ionization voltage was 70 eV
(electron impact ionization), and the ion source and interface temper-
atures were kept at 230 and 240 °C, respectively. The scan range was
set from m/z 45 to 350. The data evaluation was performed using
Xcalibur (version 1.4, Thermo Electron) software.
Kinetic Data. Unsaturated hydroxy-3(2H)-furanones (0.14-5.7 mM
final concentration) were added to an assay mixture containing 1.5 µg
of purified enzyme, 12 mM NADH in 250 µL of Na2HPO4/NaH2PO4
buffer (0.1 M, pH 7) at 30 °C for 35 min. Products were quantified by
HPLC-ESI-MSn analysis. Km and Vmax values were determined by fitting
the data to the Hanes equation using the regression program Hyper32
(version 1.00; http://homepage.ntlworld.com/john.easterby/hyper32.html).
All data points were averages of duplicates.
RESULTS
Synthesis of Substrates. Unsaturated hydroxyfuranones were
synthesized by a Knoevenagel-type condensation of HMF with
a number of aldehydes or a ketone (Figure 1) (10, 11). One
major problem was the difficulty in controlling the synthesis,
which resulted in a high number of undesired reaction products.
Necessary purification steps were hindered by the high instability
of the target compounds (11). The R,â-unsaturated ketones are
very reactive because alcohols, thiols, and water may add to
the exocyclic double bond (12). The stability of the synthesis
products increases with the length of the side chain. Therefore,
HMMF is the most unstable substance but could be trapped
with MBA as tautomeric thioether products for characterization.
One- and two-dimensional NMR experiments (1H-, COSY,
HMQC, and HMBC) and comparison with the spectra of HDMF
and EHMF confirmed the identity of the synthesized thioethers
(Tables S1a and S1b, Supporting Information). Signals for all
four possible tautomeric thioether forms were found in the NMR
spectra, but because of their signal ratios of 3:2.5:1:1 and the
low signal abundance only the carbons and hydrogens of the
two major isomers (2′-{[(3-hydroxy-2-methyl-4-oxo-4,5-dihy-
drofuran-5-yl)methyl]-sulfanyl}benzoic acid (HMMF-MBA 1)
and 2′-{[(4-hydroxy-2-methyl-3-oxo-2,3-dihydrofuran-5-yl)-
methyl]-sufanyl}benzoic acid) (HMMF-MBA 2)) could be
reliably assigned. Separation of the four isomers was not
achieved. No coupling between the aromatic and the furanone
part of the molecules was observed, but comparison of the signal
area ratios enabled the assignment of the signals to definite
molecule structures. Eight hydrogen signals were acquired for
the major HMMF-MBA 1 isomer: four downfield shifted
signals resulting from the aromatic system and four in a chemical
shift range of 2.3-4.7 ppm representing the furanone moiety.
The characteristic double-doublet signals at 3.35 and 3.65 ppm
were tentatively assigned to the hydrogen atoms bound to C-6
in HMMF-MBA 1. The signals of the aromatic part of the
second major HMMF-MBA isomer were almost identical to
those of the first isomer, but the signals of the furanone moiety
differed significantly. The singlet signal at 2.1 ppm was
tentatively assigned to the hydrogen atoms bound to C-6. The
proposed structure for HMMF-MBA 2 was further confirmed
by the doublet signal at 5.0 ppm caused by the hydrogen atom
bound to C-2 and the quartet signal for the methyl atoms
appearing at 1.4 ppm in contrast to a singlet signal at 2.3 ppm
as was observed for HMMF-MBA 1.
Trapping of HMMF in Fruit. One to two milliliters of a 70%
ethanolic solution containing 0.3 M MBA was injected into fruits on
two consecutive days. Fruits injected with only a 70% ethanolic solution
served as control.
Tracer Study. Twenty-one milligrams of D-[6-13C]-glucose dissolved
in 100 µL of water was injected into strawberry fruits in the turning
ripening stage on two consecutive days. On the third day, 50 µL of a
0.6 M MBA solution was injected into the fruits. In total, fruits were
stored for 3 days at room temperature before workup.
Isolation of the HMMF Thioether Adduct. Fruits were cut into
small pieces and immediately frozen at -20 °C. Thawed fruits (10-
20 g) were homogenized in an equal amount of water and centrifuged
(10 min at 5000g), and the supernatant was applied onto a precondi-
tioned XAD (adsorbent resin) column (30 cm × 1.5 cm). Polar
compounds were removed with 100 mL of water, nonpolar substances
were eluted with 100 mL of diethyl ether, and the semipolar metabolites
were obtained by elution with 100 mL of methanol. The diethyl ether
and methanol extracts were concentrated and analyzed by HPLC-ESI-
MSn.
Preparative RP18-HPLC-UV. A Knauer (Berlin, Germany) HPLC
system equipped with a variable wavelength detector set at 280 nm
and a preparative RP18 column (Synergi 4 µm Fusion, 250 × 15 mm,
Phenomenex, Aschaffenburg, Germany) were used. Purifications were
performed using a binary gradient starting from 100% A (H2O with
0.1% HCOOH) and 0% B (ACN with 0.1% HCOOH) increasing to
20% B in 10 min and then to 100% B during the following 10 min at
a flow rate of 5 mL/min.
HPLC-ESI-MSn. HPLC-ESI-MSn was performed using an Esquire
3000plus system equipped with an ESI interface (Bruker Daltonics,
Bremen, Germany) and an Agilent 110 HPLC system (Agilent,
Waldbronn, Germany). The separation was carried out on a Luna RP18
column (100 A, 150 × 200 mm, 5 µm, Phenomenex). The LC gradient
proceeded from 0% ACN and 100% water acidified with 0.05%
HCOOH to 20% ACN and 80% acidic water in 10 min, in 10 min to
100% ACN, 2.5 min at these conditions, then back to 100% water and
0% ACN in 5 min at a flow rate of 0.2 mL/min. The detection
wavelength was 280 nm. Mass spectra were acquired in the positive
mode. The ESI nebulizer used nitrogen at a pressure of 30 psi, and
nitrogen was used as dry gas with a flow of 9 L/min at a temperature
of 330 °C. The scan range included the masses from 20 to 500 m/z,
and the settings of the ion trap were optimized for each compound
(smart parameter settings, Bruker Daltonics). Product ion scanning was
performed with helium (3.56 × 10-6 bar) as collision gas and a collision
energy of 1.0 V. Data acquisition and evaluation was conducted with
Esquire 5.1 and DataAnalysis software (version 3.1) (Bruker Daltonics).
Because (2E)-2-ethylidene-4-hydroxy-5-methyl-3(2H)-fura-
none (EDHMF), (2E)-4-hydroxy-5-methyl-2-propylidene-3(2H)-
furanone (HMPDF), (2E)-2-butylidene-4-hydroxy-5-methyl-
3(2H)-furanone (BDHMF), and 4-hydroxy-5-methyl-2-(1-
methylethylidene)-3(2H)-furanone (HMMEF) (Figure 1) are
much more stable in a nonpolar milieu than HMMF and their
syntheses yielded significantly more product than the HMMF
synthesis, these hydroxyfuranones could be analyzed by NMR
(1H-, COSY, HMQC, and HMBC) without prior derivatization
(Tables S2a-S2c, Supporting Information) (11). Tautomeric
forms were not observed. The spectra of all molecules showed
a singlet at 2.3 ppm for the CH3-group at position C-1. Long-
range 1H, 13C couplings were observed in the HMBC spectrum
Nuclear Magnetic Resonance Spectroscopy. 1H NMR spectra were
recorded at 500.13 MHz with a Bruker DRX 500 spectrometer (Bruker,
Karlsruhe, Germany). The compounds were dissolved in either deu-
terated chloroform, acetone, or methanol. The chemical shifts were
referred to the solvent signal. The one-dimensional and two-dimensional
COSY, HMQC, and HMBC spectra were acquired and processed with
standard Bruker software (XWIN-NMR).
HRGC-MS. High-resolution gas chromatography mass spectrometry
was performed with a Trace GC 2000 Ultra (Thermo Electron Corp.,
Dreieich, Germany) connected to a quadrupole (Trace DSQ) mass
spectrometer and an autoinjector AI 3000. The GC was equipped with
a split injector (1:20) and fitted with a DB-FFAP fused silica capillary
column (30 m × 0.32 mm inner diameter; thickness of the film )

6708 J. Agric. Food Chem., Vol. 55, No. 16, 2007
Klein et al.
between the methyl hydrogen atoms and the C-2 and C-3 carbon
atoms of the dihydrofuranone ring system, respectively. By
elongation of the carbon chain at the exocyclic double bond,
the signals for the hydrogens and carbons of the CH2 groups at
positions C-7 and C-8 shifted high field compared with the
signals of the CH3 group. The structures of the side chains were
1
1
also confirmed by H, H spin couplings of the neighboring
hydrogen atoms at positions 6-9.
Enzymatic Conversion. For substrate screening, we used the
synthesized compounds (HMMF, EDHMF, HMPDF, BDHMF,
HMMEF) and diverse naturally occurring and non-natural R,â-
unsaturated carbonyl compounds (2E-hexenal, 3E-hexen-2-one,
5-methyl-3E-hexen-2-one, cyclohexa-1,2-dione, buta-2,3-dione,
coumarin, astaxanthin, cinnamon aldehyde, p-coumaric acid,
ferulic acid, dimethoxycinnamic acid, benzil) together with
recombinant FaEO, produced by FaEO expressing E. coli and
NADH and analyzed the products by HRGC-MS and HPLC-
ESI-MSn. Control experiments were performed with protein
fractions from E. coli cells transformed with the empty vector.
Only the newly synthesized compounds HMMF, EDHMF,
HMPDF, and BDHMF except HMMEF were converted to their
saturated derivatives. The enzymatic products HDMF and
EHMF were identified by their electrospray ionization and
electron impact mass spectra in comparison with authentic
reference material (Figures S1 and S2, Supporting Information).
The structures of HMPF and BHMF were tentatively assigned
because of their fragmentation pattern (Figures S1 and S2,
Supporting Information).
Figure 2. HPLC-ESI-MSn analysis of the HMMF thioether product. Product
ion spectrum of m/z (+) 281 of the synthesized reference compound (A),
of the compound isolated from pineapple after treatment with MBA (B),
and isolated from tomato treated with MBA (C). Extracted ion chromato-
gram m/z (+) 281 of extracts obtained from control fruits (D) and
strawberries after the injection of MBA into fruits of the green (E), pink
(F), and red (G) ripening stage. Fruits were treated for 2 days with 0.6 M
ethanolic MBA solution. After freezing and thawing, the fruits were
processed by a XAD solid-phase column, and the obtained diethyl ether
fraction was analyzed by HPLC-ESI-MSn under the same conditions.
To calculate the Km and kcat values, we used EDHMF,
HMPDF, and BDHMF in concentrations ranging from 0.14 to
5.7 mM with an excess of NADH (12 mM). HMMF was too
unstable to obtain reliable values. The amounts of the product
EHMF were determined by HPLC-ESI-MSn with the help of a
calibration curve. The lack of reference material for HMPF and
BHMF forced us to use the EHMF standard curve to calculate
the amounts of HMPF and BHMF as “EHMF equivalents”,
which is justified because of the similar structure and ionization
behavior. Overall, the values fell in the same order of magnitude
traces were detected in raspberry and the concentrations in kiwi
and mango were lower than our detection limit of about 0.01
mg/kg fresh weight. The controls were devoid of the thioether
product (Figure 2D). Levels of HMMF correlated with the
ripening stage of the fruits because the thioether product was
only found in strawberry in the pink and red ripening stage but
not in green immature fruits when determined under the same
conditions (Figure 2E-G).
(Figure 1). HMPDF exhibited the highest kcat value (2.69 s-1
)
In Vivo Studies for the Detection of 13C-HMMF-MBA. It
and catalytic efficiency (kcat/Km of 2.60 mM-1 s-1), but the Km
values decreased with decreasing side chain lengths (2.14, 1.04,
and 0.82 mM).
2
had already been shown that HDMF becomes 13C- and H-
labeled when isotopically labeled glucose is injected into
ripening strawberry fruits (4, 8). Thus, to confirm HMMF as
the natural precursor of HDMF, we applied 100 µL of an
aqueous solution of 21 mg of D-[6-13C]-glucose into detached
pink strawberry fruits. After 2 days, 50 µL of a 0.6 M ethanolic
solution of MBA was injected. HPLC-ESI-MSn analysis of
extracts showed that the labeling degree of the thioether caused
by the natural abundance of 13C (determined by the M + 1
signal) increased from 13% in the untreated control fruits, up
to 33% in fruits treated with D-[6-13C]-glucose (Figure 3A,D).
The MSn experiments confirmed that the 13C was exclusively
integrated into the hydroxyfuranone moiety (Figure 3B).
Homologous Enzyme from Tomato. The demonstration of
HMMF as natural precursor of HDMF in fruit of different
species let us suppose that these fruits contain enzymes similar
to FaEO. We found a putative quinone oxidoreductase sequence
similar to FaEO in the tomato EST database (http://ted.bti.
cornell.edu/) and pineapple database (http://genet.imb.uq.edu.au/
Pineapple/) (Figure 4). The gene from tomato was cloned and
expressed in E. coli. It shows 63 and 71% identity with FaEO
on the nucleotide and amino acid levels, respectively (Table
S3, Supporting Information). It is also predicted to contain a
chloroplast transit peptide at the N-terminus unlike FaEO (13).
The recombinant enzyme lacking the signal peptide accepted
In Vivo Studies for the Detection of HMMF-MBA.
Although HDMF has been detected in a number of fruits, its
biosynthetic pathway has only been investigated in strawberries
until now. Because HMMF had already been identified as
precursor of HDMF in strawberry fruit after derivatization with
MBA (9), HDMF forming fruits such as tomato, raspberry,
pineapple, kiwi, and mango were treated with an ethanolic
solution of the trapping reagent (4, 7). We wanted to know
whether HDMF is formed in the different fruits by the same
pathway as already determined in strawberry. Apples that do
not produce HDMF as well as fruits injected with ethanol served
as controls. The HMMF-MBA thioether adduct was identified
by its retention time (19.1 min) and MS2 data showing
characteristic fragments at m/z 281, 263, 155, and 127 (Figure
2A). The adduct was clearly detected by HPLC-ESI-MSn in
tomatoes and pineapples (Figure 2B,C). We found traces of
the thioether in raspberry but were unable to carry out MSn
experiments because of the limited amount. The HMMF-MBA
adduct was not detected in kiwi and mango. The levels of the
thioether mirrored the levels of HDMF because GC-MS
analysis showed that tomato and pineapple contained appreciable
amounts of HDMF (>0.1 mg/kg fresh weight), whereas only

Enone Oxidoreductases
J. Agric. Food Chem., Vol. 55, No. 16, 2007 6709
Figure 3. HPLC-ESI-MSn analysis of the HMMF thioether product isolated
from strawberry fruits treated with MBA. Sections of the product ion mass
+
spectra showing the pseudo molecular ion [M
+
H] at m/z 281 (A, D),
+
the [M-MBA
in the molecule (B, E), and the [MBA
treated with
+
H] ion at m/z 127 representing the hydroxyfuranone moiety
H] ion (C, F). Strawberries were
-[6-13C]-glucose and MBA (A, B, C) or only with MBA (control)
+
+
D
(D, E, F). After freezing and thawing, the fruits were processed by a XAD
solid-phase column, and the obtained diethyl ether fraction was analyzed
by HPLC-ESI-MSn.
HMMF, EDHMF, HMPDF, and BDHMF as substrates and
reduced them to their saturated derivatives HDMF, EHMF,
HMPF, and BHMF, respectively (Figures 1 and S3, Supporting
Information). HMMEF and straight-chain substrates were not
accepted as observed for FaEO. SlEO showed kcat values (0.15-
0.39 s-1) generally lower than those of FaEO (0.82-2.14 s-1),
by a factor of 5-10, but similar Km values (1.13-2.41 mM,
respectively, 0.82-2.14 mM). Overall, the substrates were less
efficiently reduced by the tomato enzyme with a slight prefer-
ence for EDHMF. We designate this protein as SlEO (Solanum
lycopersicon enone oxidoreductase) because of the catalyzed
reaction.
Figure 4. Amino acid sequence comparison of the strawberry FaEO
protein and putative quinone oxidoreductases from higher plants and E.
coli. The sequences were aligned using the ClustalW program. Consensus
amino acids are shaded. The NAD(P)H binding motif G(A)XXGXXG is
marked. The accession numbers are AY048861 for Fragaria x ananassa
(FaEO), AJ001445 for Fragaria vesca (FvEO), TC124720 for Solanum
lycopersicon (SlEO), JBW022G03 for Ananasa comosa (AcQR), AF451799
for Arabidopsis thaliana (AtQR), Q39172 for Arabidopsis thaliana (AtAER),
and AAC43145 for Escherichia coli (EcQR).
DISCUSSION
Until now, only little was known about the physiological roles
of NAD(P)H-dependent quinone oxidoreductases in plants.
During a study to uncover genes that perform antioxidant
functions, a gene encoding an NADPH/quinone oxidoreductase-
like protein was isolated from Arabidopsis thaliana (14) (Figure
4). The recombinant protein expressed in E. coli showed an
NADPH-dependent oxidoreductase activity to reduce quinones
(15). Upon a search for more physiologically relevant substrates,
cytotoxic 2-alkenals and oxenes were identified as potential in
vivo substrates (16). The R,â-unsaturated bond of 2-alkenals
was specifically reduced, but the aldehyde group was retained.
On the basis of this unique activity, the name alkenal reductase
(AER) has been recommended. Only two proteins have been
reported to have AER activity, AER from Arabidopsis (AtAER)
and leukotriene B4 12-hydroxy dehydrogenase from rat (17).
The AER-catalyzed reaction significantly lowers the toxicity
of reactive carbonyls because the electrophilicity of the R-car-
bon, which reacts with target molecules, is diminished by the
reduction of the unsaturated bond.
In this study, we show that FaEO reduces the double bond
adjacent to the carbonyl group in HMMF, EDHMF, HMPDF,
and BDHMF similar to AER although FaEO was putatively
assigned earlier as quinone oxidoreductase (Figure 1). HMMEF
was not accepted probably because of the second methyl group,
which causes steric hindrance. Accurate kinetic data were
obtained for all substrates except HMMF, which was too
unstable in aqueous solution. However, the conversion of
HMMF to HDMF was clearly demonstrated by HPLC-ESI-MSn
analysis (9). Because the Km and kcat values for the furanone
substrates fell in the same range, we assume similar data for
HMMF (Figure 1). The specificity (kcat/Km) ranged from 1.4
to 2.7 mM-1 s-1. These values differ significantly from the
values obtained with 9,10-phenanthrene quinone (PQ) (Km
)
12 µM, kcat/Km ) 650 mM-1 s-1), a synthetic substrate that is
accepted by FaEO and also by AtAER (Km ) 0.65 µM, kcat/Km
) 151 mM-1 s-1) (9, 15). However, in contrast to FaEO, the

6710 J. Agric. Food Chem., Vol. 55, No. 16, 2007
Klein et al.
alkenal reductase from A. thaliana catalyzes the reduction of a
number of 1,4-quinones (15). A recently crystallized E. coli
quinone oxidoreductase (EcQR) also catalyzes the NAD(P)H-
dependent reduction of quinone substrates but shows only 25
and 25% identity with FaEO and AtAER (18) (Figure 4 and
Table S3, Supporting Information).
HDMF. EDHMF, HMPDF, and BDHMF have not been found
in nature up to now. Although HMMF is very reactive and
unstable, it is the precursor of HDMF in fruits. This implies
that the supposed, but not yet characterized, enzyme that forms
HMMF in vivo must either strongly protect its product or
immediately transfer it to the detoxifying EOs after its produc-
tion. Otherwise, HMMF could cause severe damage as it might
react with essential components of the cell. Therefore, FaEO
and SlEO probably perform, similarly to AtAER, a detoxifica-
tion reaction (15, 16).
This article demonstrates that not only strawberry but also
tomato, and likely other fruit such as pineapple, express an
enzyme that converts HMMF into HDMF, which is an important
flavor compound. It also shows that the substrate specificity of
both tomato and strawberry enzymes is toward hydroxyfura-
nones. Thus, the data provide the foundation for the biotech-
nological improvement of the aroma composition of agronomi-
cally relevant fruits.
EHMF, the product formed by FaEO from EDHMF, is an
important constituent of soy sauce and cheese and determines
their aromas. In soy sauce, it is assumed that EHMF is formed
by the yeasts from an unknown precursor that is produced by
the amino-carbonyl reaction of a pentose with an amino acid
during heating (19). As there are NAD(P)H-dependent oxi-
doreductases known in yeasts, it is possible that EDHMF plays
a role in the biosynthesis of EHMF during soy sauce preparation
(20). The substrates HMPDF and BDHMF as well as the
resulting enzymatic products HMPF and BHMF have not yet
been described.
Recently, the presence of HMMF in ripening strawberry fruits
was demonstrated by trapping the unstable R,â-unsaturated
carbonyl compound with MBA as thioether adduct (9). Here,
we prove that HMMF is also produced by tomato, pineapple,
and raspberry fruits that contain appreciable amounts (>0.1 mg/
kg) of HDMF. Because only minor levels of HDMF have been
found in mangoes and kiwis, it is possible that we could not
detect HMMF in these fruits. The highest levels of HMMF were
detected in fully ripe, red fruits consistent with the ripening-
induced expression of FaEO and the accumulation of HDMF
during the late stages of strawberry ripening (Figure 2). Feeding
experiments with D-[6-13C]-glucose in strawberry fruits have
already demonstrated the incorporation of the labeled carbon
into HDMF (8). In line with the assumption that HMMF is the
precursor of HDMF, our results show that also HMMF becomes
13C-labeled when D-[6-13C]-glucose is injected into ripening
strawberry fruits. Together, these data suggest that HDMF is
synthesized in the different fruit species by a similar pathway.
ABBREVIATIONS USED
AtAER, Arabidopsis thaliana alkenal reductase; BDHMF,
(2E)-2-butylidene-4-hydroxy-5-methyl-3(2H)-furanone; BHMF,
2-butyl-4-hydroxy-5-methyl-3(2H)-furanone; EDHMF, (2E)-2-
ethylidene-4-hydroxy-5-methyl-3(2H)-furanone; EHMF, 2-ethyl-
4-hydroxy-5-methyl-3(2H)-furanone; FaEO, Fragaria x anan-
assa enone oxidoreductase; FaQR, Fragaria x ananassa quinone
oxidoreductase; HDMF, 4-hydroxy-2,5-dimethyl-3(2H)-fura-
none; HIMF, 4-hydroxy-2-isopropyl-5-methyl-3(2H)-furanone;
HMF, 4-hydroxy-5-methyl-3(2H)-furanone; HMMEF, 4-hy-
droxy-5-methyl-2-(1-methylethylidene)-3(2H)-furanone; HMMF,
4-hydroxy-5-methyl-2-methylene-3(2H)-furanone; HMPDF, (2E)-
4-hydroxy-5-methyl-2-propylidene-3(2H)-furanone; HMPF, 4-hy-
droxy-5-methyl-2-propyl-3(2H)-furanone; HRGC-MS, high-
resolution gas chromatography mass spectrometry; SlEO,
Solanum lycopersicon enone oxidoreductase; MBA, 3-mercap-
tobenzoic acid; NMR, nuclear magnetic resonance spectroscopy;
XAD, adsorbent resin; HPLC-ESI-MSn, high-performance liquid
chromatography electrospray ionization multidimensional mass
spectrometry.
Therefore, we screened cDNA databases for sequences similar
to FaEO and found a full-length cDNA in the tomato expression
library (http://ted.bti.cornell.edu/) and a partial sequence in the
pineapple cDNA database (http://genet.imb.uq.edu.au/Pineapple/).
FaEO shows 71 and 73% identity with the protein sequence
from tomato (SlEO) and pineapple (AcQR), respectively (Figure
4 and Table S3, Supporting Information). SlEO carries a
putative transit peptide of about 60 amino acids at the N-
terminus that probably directs the protein into chloroplasts
(Figure 4). A signal peptide has not been found for FaQR. But
since D-fructose-1,6-diphosphate, the starting material for HDMF
biosynthesis, is present in chloroplasts and the cytosol, it is
conceivable that HDMF formation proceeds in both compart-
ments. We were able to clone SlEO and functionally express
the protein in E. coli. The purified enzyme reduced HMMF,
EDHMF, HMPDF, and BDHMF with slightly lesser efficiency
than FaEO (Figure 1). Thus, it seems that SlEO is involved in
the biosynthesis of HDMF in tomato fruit. A putative QR protein
sequence from A. thaliana (AtQR) was also found by BLAST
search showing 69 and 70% identity with FaEO and SlEO,
respectively, although AtAER displays only 27 and 28% identity
with these proteins. The function of AtQR remains to be
clarified.
ACKNOWLEDGMENT
We thank Yu Fan Yang and Regina Dick for technical support
and Heather Coiner and Mark Somoza for correcting the
manuscript.
Supporting Information Available: Table S1a: NMR signal
assignments of HMMF-MBA 1. Table S1b: NMR signal
assignments of HMMF-MBA 2. Table S2a: 1H NMR signal
assignments of BDHMF. Table S2b: 1H NMR signal assign-
ments of HMPDF. Table S2c: 1H NMR signal assignments of
EDHMF. Table S3: Amino acid identities calculated by
ClustalW of protein sequences for proven and putative enone
and quinone oxidoreductases. Figure S1: HPLC-ESI-MSn
analysis of FaEO substrates and corresponding products. Figure
S2: GC-MS analysis of FaEO substrates and corresponding
products. Figure S3: SDS-PAGE analysis of purified recom-
binant FaEO and SlEO. This material is available free of charge
via the Internet at http://pubs.acs.org.
The major in vivo function of the enone oxidoreductases from
strawberry and tomato is the conversion of HMMF to HDMF
as demonstrated by the activity-guided isolation of FaEO (9),
the conversion of HMMF by recombinant FaEO and SlEO, the
detection of HMMF in ripening strawberry and tomato fruit,
and the conversion of labeled glucose to HMMF and finally to
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Received for review April 11, 2007. Revised manuscript received May,
31, 2007. Accepted May 31, 2007. This work was supported by Degussa
AG.
JF071055O