Download A New Type of a Multifunctional ß-Oxidation

A New Type of a Multifunctional ␤-Oxidation
Enzyme in Euglena
Uwe Winkler*, Werner Säftel, and Helmut Stabenau
Department of Biology, University of Oldenburg, P.O. Box 2503, D–26111 Oldenburg, Germany
The biochemical and molecular properties of the ␤-oxidation enzymes from algae have not been investigated yet. The present
study provides such data for the phylogenetically old alga Euglena (Euglena gracilis). A novel multifunctional ␤-oxidation
complex was purified to homogeneity by ammonium sulfate precipitation, density gradient centrifugation, and ion-exchange
chromatography. Monospecific antibodies used in immunocytochemical experiments revealed that the enzyme is located in
mitochondria. The enzyme complex is composed of 3-hydroxyacyl-coenzyme A (-CoA) dehydrogenase, 2-enoyl-CoA hydratase, thiolase, and epimerase activities. The purified enzyme exhibits a native molecular mass of about 460 kD, consisting
of 45.5-, 44.5-, 34-, and 32-kD subunits. Subunits dissociated from the complete complex revealed that the hydratase and the
thiolase functions are located on the large subunits, whereas two dehydrogenase functions are located on the two smaller
subunits. Epimerase activity was only measurable in the complete enzyme complex. From the use of stereoisomers and
sequence data, it was concluded that the 2-enoyl-CoA hydratase catalyzes the formation of l-hydroxyacyl CoA isomers and
that both of the different 3-hydroxyacyl-CoA dehydrogenase functions on the 32- and 34-kD subunits are specific to l-isomers
as substrates, respectively. All of these data suggest that the Euglena enzyme belongs to the family of ␤-oxidation enzymes that
degrade acyl-CoAs via l-isomers and that it is composed of subunits comparable with subunits of monofunctional ␤-oxidation
enzymes. It is concluded that the Euglena enzyme phylogenetically developed from monospecific enzymes in archeons by
non-covalent combination of subunits and presents an additional line for the evolutionary development of multifunctional
␤-oxidation enzymes.
The degradation of activated fatty acids by
␤-oxidation principally requires four different enzymecatalyzed reactions. The acyl-CoA esters are desaturated in an initial reaction. The resulting 2-enoyl-CoA
esters are then hydrated to 3-hydroxyacyl-CoA esters,
which in a third step are dehydrogenated concomitant
with the reduction of NAD. The fourth reaction is the
acetyl-CoA yielding thiolytic cleavage of the
3-ketoacyl-CoA esters formed in the dehydrogenation
process. Because the fatty acid chains are reduced by
only two carbon atoms in the course of these successive
reactions, the reaction sequence has to be repeated until
the fatty acids have been completely degraded (Fig. 1).
The first reaction, in which acyl-CoA esters are desaturated can be catalyzed by different types of enzymes, either an acyl-CoA oxidase transferring electrons to molecular oxygen and thereby producing H2O2
or an acyl-CoA dehydrogenase coupled—via an electron transferring protein—with an electron transport
chain reducing oxygen to H2O, but incapable of forming H2O2. So far, the acyl CoA oxidase has been detected in microbodies of higher plants (Kindl, 1993),
animals (Hashimoto, 1987), and some fungi (Kunau et
al., 1987). Acyl-CoA dehydrogenase, however, is localized in bacteria (Klein, 1973), the mitochondria of animals (Hall, 1978), and primitive algae (Stabenau, 1992),
but it was also found in the microbodies of developed
* Corresponding author; e-mail [email protected];
fax 49 – 411–798 –3331.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.013151.
algae (Winkler et al., 1988) and in the fungus Neurospora
crassa (Kionka and Kunau, 1985). The existence of biochemical pathways without an H2O2 metabolism may
be characteristic of microbodies in phylogenetically old
organisms (Stabenau, 1992; Kunau et al., 1995).
In the following steps, as in the first reaction, there
are different enzyme types catalyzing the hydration
and dehydrogenation reactions. In particular, they differ in stereospecificity of substrates required and products formed. All in all, the different enzymes of both
reaction steps are assigned to two different metabolic
routes. The first and most widespread route consists of
a 2-enoyl-CoA hydratase, also termed enoyl-CoA hydratase 1 or crotonase. It hydrates 2-trans-enoyl-CoA
esters to l-3-hydroxyacyl-CoA esters, but also 3-cisenoyl-CoA esters to d-3-hydroxyacyl-CoA esters. Because only l-dependent 3-hydroxyacyl-CoA dehydrogenases are available for the subsequent reaction step in
this first metabolic route, d-3-hydroxyacyl-CoA esters
must be transformed into the corresponding l-form in
case they arise, for example, during metabolism of unsaturated fatty acids. This transformation takes place by
means of an epimerase reaction.
The second metabolic route is characterized by a
2-enoyl-CoA hydratase that hydrates 2-trans-enoylCoA to d-3-hydroxyacyl-CoA esters. It is called
d-trans-enoyl-CoA hydratase, enoyl-CoA hydratase
2, or novel enoyl-CoA hydratase. The resulting d-3hydroxyacyl-CoA esters are further metabolized by
d-3-hydroxyacyl-CoA dehydrogenases.
Enzymes of the first route are monospecific proteins, which are characteristic of animal mitochon-
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753
Winkler et al.
Figure 1. Most common pathways for the degradation of fatty acid CoA esters by ␤-oxidation enzymes in the different
organisms. In the first reaction of the ␤-oxidation cycle, acyl-CoA esters are desaturated to ⌬2-trans enoyl-CoA esters by acyl
CoA oxidases or acyl CoA dehydrogenases. Oxidases are located in microbodies of higher plants and animal tissue.
Dehydrogenases are found in bacteria, animal mitochondria, and microbodies of some fungi and in algae. For the following
reaction of the ␤-oxidation cycle, different pathways are used. Pathway I, The ⌬2-trans enoyl-CoA esters are metabolized by
monofunctional crotonases (also named ECH-1), L-hydroxyacyl-CoA dehydrogenases, and thiolases. This pathway is located
in bacteria and animal mitochondria. Pathway II, Crotonase, and L-hydroxyacyl-CoA dehydrogenase are domains of a
common polypeptide that also metabolizes ⌬2-cis-enoyl-CoA esters occurring in unsaturated fatty acids. These ⌬2-cis-enoylCoA esters are also suitable substrates for the crotonase, but the resulting product is D-hydroxyacyl-CoA, which is
transformed to the L-form by an epimerase function of the polypeptide. This MFP is specifically found in microbodies of
higher plants and animals. Pathway III, The polypeptide that carries functions for crotonase, L-hydroxyacyl-CoA dehydrogenase, and epimerase is combined with a second polypeptide that displays thiolase activity. This type of MFP is found in
bacteria and animal mitochondria. Pathway IV, The ⌬2-trans enoyl-CoA esters are metabolized by a D-specific 2-enoyl-CoA
hydratase (D-ECH; ECH-2) that forms D-specific products and a D-hydroxyacyl-CoA dehydrogenase that is specific to
D-hydroxyacyl-CoA esters as substrates. Both enzyme functions are located at a common polypeptide. This bifunctional
protein is located in microbodies of fungi and in rat liver tissue.
dria (Osumi and Hashimoto, 1980; Fong and Schulz,
1981) but have also been detected in some bacteria
(O‘Connell et al., 1990; Klenk et al., 1997). Hydratase
and dehydrogenase functions, however, may also be
localized on a common polypeptide that most likely
resulted from a fusion process between the genes of
both monospecific enzymes during evolution (Kamijo et al., 1993). Corresponding multifunctional proteins (MFPs) are the multifunctional enzyme 1 in
animal peroxisomes (Furuta et al., 1980), the trifunctional and tetrafunctional proteins from plant glyoxysomes (Kindl, 1993), the ␣-subunit of the trifunctional
754
protein from rat liver mitochondria (Uchida et al.,
1992), and the ␣-subunit of the multifunctional fatty
acid oxidation complex from Escherichia coli (Binstock et
al., 1977) and other eubacteria (Kunau et al., 1995). The
MFPs from rat liver mitochondria and eubacteria additionally contain ␤-subunits with thiolase function.
Hydratases and dehydrogenases of the second metabolic route are always domains of a common polypeptide. So far, such bifunctional enzymes have only been
detected in peroxisomes, like the multifunctional enzyme 2 from rat (Dieuaide-Noubhani et al., 1997) and
the ␤-oxidation enzymes found in different fungi
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Plant Physiol. Vol. 131, 2003
Multifunctional ␤-Oxidation Enzyme in Euglena
RESULTS
Enzymes of the Fatty Acid ␤-Oxidation
Pathway in Euglena
Figure 2. Increase in ␤-oxidation enzyme activity in Euglena. Autotrophically grown cells were transferred to a growth medium containing 5 mmol L⫺1 hexanoic acid as sole carbon source and cultured
in the dark for 6 d. Enzyme activities for 3-hydroxyacyl-CoA dehydrogenase (Dehydrogenase), 2-enoyl-CoA hydratase (Hydratase),
and thiolase were measured in the crude homogenates and are
expressed as nanomoles per minute per 106 cells. Growth is indicated by the cell number on the right ordinate.
(Thieringer and Kunau, 1991). All d-enoyl-CoA hydratase and all d-3-hydroxyacyl-CoA dehydrogenase
domains from the different organisms exhibit pronounced amino acid sequence similarities among themselves (Qin et al., 1997). The same is true for monospecific enzymes or domains displaying 2-enoyl-CoA
hydratase 1 or l-3-hydroxyacyl-CoA dehydrogenases
activity, respectively (Baldwin, 1993; Kamijo et al.,
1993). Because there are no sequence relations between
the d-specific bifunctional enzymes of the second route
and the l-specific ␤-oxidation enzymes of the first one
(Hiltunen et al., 1992), the enzymes of both routes obviously belong to different evolutionary families.
Enzymes of all four reactions of the ␤-oxidation
pathway have also been detected in algae. In the
group of Heterokontophyta, corresponding enzyme
activities have only been found in the mitochondria
(Gross et al., 1985; Winkler and Stabenau, 1994). In
the green alga Chara spp., however, they are exclusively localized in peroxisomes (H. Stabenau, U.
Winkler, and W. Säftel, unpublished data). A localization in mitochondria as well as in peroxisomes
was found in the green algae Mougeotia spp. and
Eremosphaera spp. (Stabenau et al., 1984; Winkler et
al., 1988) and in heterotrophically grown Euglena
(Euglena gracilis; Graves and Becker, 1974).
The biochemical and molecular properties of the
␤-oxidation enzymes from algae have not been investigated yet. Hence, the present study provides such
data for the first time, to our knowledge. We chose
the alga Euglena for our investigations because it is
one of the few algal species that can be cultured
heterotrophically on fatty acids (Hosotani et al.,
1988). Moreover, Euglena is one of the oldest eukaryotes (Tessier et al., 1997) and may therefore be a
useful organism to enhance our knowledge about the
evolution of ␤-oxidation enzymes.
Plant Physiol. Vol. 131, 2003
In Euglena, an increase in the activity of the enzymes involved in the ␤-oxidation pathway was
measured after the conditions were changed from
autotrophic to heterotrophic growth with hexanoic
acid as the sole source of carbon and energy (Fig. 2).
Enzyme activity reached a maximum after 2 d of
heterotrophic growth. Cells were harvested at this
time and used for the experiments described here.
Enzymes of ␤-oxidation in the crude homogenate of
hexanoic acid-grown algae were separated according
to their molecular mass by Suc gradient centrifugation. As shown in Figure 3A, the distribution patterns
of 2-enoyl-CoA hydratase and thiolase each show one
peak in fraction eight, indicating a molecular mass of
around 146 kD. Thiolase and 2-enoyl-CoA hydratase
having similar molecular masses have already been
described (Staak et al., 1978; Fong and Schulz, 1981).
The distribution of the 3-hydroxyacyl-CoA dehydrogenase reveals two peaks. In fraction four, the enzyme
protein should possess a molecular mass of around 41
kD, which makes it comparable with monospecific
l-3-hydroxyacyl-CoA dehydrogenase from animal mitochondria (Osumi and Hashimoto, 1980). The molecular mass of the enzyme in fractions 11 to 12 is expected to be somewhere between 390 and 540 kD, yet
no comparable 3-hy-droxyacyl-CoA dehydrogenase
has been found in other organisms. That is why we
have directed our attention to this enzyme. The biochemical and molecular properties of the protein associated with the 3-hydroxyacyl-CoA dehydrogenase
activity will be detailed in the following section.
Purification of 3-Hydroxyacyl-CoA
Dehydrogenase from Euglena
Enzyme purification was performed by successive
ammonium sulfate (AS) precipitation, Suc gradient
centrifugation, and ion-exchange chromatography on
sulfopropyl- and dimethylaminoethyl columns (Table
I). The highest 3-hydroxyacyl-CoA dehydrogenase activity was obtained in the fraction of 52.5% to 62.5%
AS. Most of the proteins precipitating together with
this activity were removed by Suc density centrifugation. The main peak of 3-hydroxyacyl-CoA dehydrogenase appeared in fraction 11 of the gradient (Fig.
4A). In accordance with the enzyme activity profile in
the SDS gel of fractions 10 to 12, three bands of 45, 34,
and 32 kD were found (Fig. 4B). Further purification
was obtained using cation-exchange chromatography
on sulfopropyl columns. The 3-hydroxyacyl-CoA dehydrogenase was not bound to this column, but contaminating proteins with ␤-oxidation activity were removed. Final purification of the enzyme was achieved
by means of dimethylaminoethyl anion-exchange
chromatography (Fig. 5). The 3-hydroxyacyl-CoA de-
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755
Winkler et al.
four different subunits. In Table II, some data are
presented characterizing the enzyme, which was apparently purified to homogeneity after dimethylaminoethyl anion-exchange chromatography.
Distribution of Enzyme Functions on the Subunits
The molecular structure of the Euglena MFP proved
to be unstable under certain experimental conditions.
Complete dissociation into protomers was obtained by
applying low temperatures, high concentration of
some salts and homogenization procedures like sonification, suggesting that the subunits are combined
through non-covalent interactions. Graduated dissociation, by which the smaller 32- and 34-kD subunits
were obtained, was observed after eluting the MFP
from the dimethylaminoethyl column (Fig. 5B-I). In
the fractions containing dissociated 32- and 34-kD
subunits only 3-hydroxyacyl-CoA dehydrogenase activity could be measured. From analogous experiments with anion-exchange columns resulting in partial dissociation of the 45-kD subunits, it was
concluded that the 2-enoyl-CoA hydratase and thiolase functions are located on 45-kD subunits (data not
shown). Activity for epimerase was only measured in
the native MFP.
Figure 3. Distribution of ␤-oxidation enzymes from hexanoic acidgrown Euglena in a Suc gradient. Proteins of a crude homogenate
were separated according to their molecular masses in a linear
gradient from 5% to 20% (w/w) Suc. Centrifugation was performed at
140,000g for 18 h and at 20°C. A, Distribution of enzyme activities
in the gradient. Enzyme activity is expressed as micromoles per
minute per milliliter fraction for 3-hydroxyacyl-CoA dehydrogenase
(Dehydrogenase) and 2-enoyl-CoA hydratase (Hydratase). Full-scale
activity for the thiolase reaction corresponds to 50 nmol min⫺1 mL⫺1
fraction. The distribution of molecular masses in the fractions was
obtained from the distribution of standard proteins. B, Analysis of the
proteins distributed in the fractions from the gradient by SDS-PAGE
and Coomassie staining. Proteins were separated in a 5% to 15%
(w/v) gradient gel. Lane M, Molecular mass markers as indicated on
left. The arrows indicate the protein bands that correlate with the
main peak of activity for 3-hydroxyacyl-CoA dehydrogenase in fractions 10 to 11.
hydrogenase coeluated with activity of 2-enoyl-CoA
hydratase, thiolase, and epimerase. The activity profile of all enzymatic reactions agreed with the abundance of three proteins bands of 45, 34, and 32 kD in
an SDS gel (Fig. 5B-I). As demonstrated in Figure
5B-III, fraction 7 of the eluate only contained one
native protein with activity for all four enzymatic
reactions, which are summarized in Table II. In an SDS
gel it could be shown that the native protein is composed of subunits with 45, 34, and 32 kD (Fig. 5B-IV).
Using an improved gel system, however, we could
separate the protein in the 45-kD band into two peptides with only minor differences in their molecular
masses (Fig. 5B-II). Thus, the MFP actually consists of
756
Stereospecificity of the 2-Enoyl-CoA
Hydratase Reaction
Due to its dehydrating activity, the 2-enoyl-CoA
hydratase from Euglena is capable of catalyzing both
the hydratase reaction and the reverse reaction. To
determine the stereospecificity of the enzyme, we
have measured the dehydratase activity of the purified MFP by using either the R(l) or S(d) isomers of
3-hydroxy-3-phenylpropionyl-CoA as substrates
(Baes et al., 2000). Significant activity was only determined to take place with the R-isomer (Table III).
In this regard, the 2-enoyl-CoA hydratase from Euglena is similar to the mitochondrial monospecific
2-enoyl-CoA hydratase and the 2-enoyl-CoA hydratase function of the peroxisomal multifunctional
enzyme 1 but dissimilar to peroxisomal multifunctional enzyme 2, which is exclusively specific to S(d)isomers (Mao et al., 1994).
Protein Microsequencing and Search for Homologies
Upon analyzing the amino acid sequence, only the
32- and 34-kD subunits yielded significant data. A
search for proteins related to the 32-kD subunit revealed that its amino acid sequence is 51% to 31%
homologous to monospecific 3-hydroxyacyl-CoA dehydrogenases and to domains for 3-hydroxyacyl-CoA
dehydrogenases of MFPs that are known to possess
l-specific dehydrogenases (Fig. 6). In addition, the
monospecific dehydrogenase from Mus musculus, producing sequence homology to the Euglena 32-kD sub-
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Plant Physiol. Vol. 131, 2003
Multifunctional ␤-Oxidation Enzyme in Euglena
Table I. Purification of 3-hydroxyacyl-CoA dehydrogenase from Euglena
Activity of 3-Hydroxyacyl-CoA Dehydrogenasea
Purification Step
Total protein
Total activity
Specific activity
Purification
Recovery
mg
␮mol min⫺1
␮mol min⫺1 mg⫺1 protein
-fold
%
177
14.5
2.2
0.8
0.3
105
24
20
16
12
0.6
2.0
9
20
40
1.0
3
15
33
67
100
27
18
14
11
Crude homogenate
AS (52.5 to 62.5%)
Suc gradient
Sulfopropyl column; void volumeb
Dimethylaminoethyl column
a
Enzyme activity was measured at the standard substrate concentration of 15 ␮mol L⫺1. The specific activity for the 3-hydroxyacyl-CoA
dehydrogenase reaction of 40 ␮mol min⫺1 mg⫺1 protein of the purified enzyme corresponds to a specific activity of 260 ␮mol min⫺1 mg⫺1
b
protein at substrate saturation.
Most of the activity was not bound to this column, but some contaminating proteins were removed.
unit, is also an l-specific enzyme (the stereospecificity
for the other monospecific enzymes was not indexed
in the databases). No similarities to domains of
d-specific 3-hydroxyacyl-CoA dehydrogenases in
MFPs were detected. Altogether, the sequence analysis and the activity measurements indicate that an
l-3-hydroxyacyl-CoA dehydrogenase function is located on the 32-kD subunit of the Euglena MFP. The
amino-terminal sequence of the 34-kD subunit shows
only limited similarities to the 32-kD subunit, but is
homologous to monospecific 3-hydroxyacyl-CoA dehydrogenases from bacteria. Therefore the 34 kD
should also carry a 3-hydr-oxyacyl-CoA dehydrogenase function. Many dehydrogenases possess a highly
conserved GxGxxG sequence that is located around
the NAD-binding site (Kamijo et al., 1993). Such
GxGxxG motives are also present in the sequences of
both subunits from the Euglena MFP (Fig. 6), providing additional evidence that they carry dehydrogenase
functions.
Cellular Localization of the Euglena MFP
Polyclonal antibodies directed against the Euglena
MFP were raised in rabbits. After immunoblot analysis, the antibodies proved to be monospecifically directed against all subunits of the MFP (Fig. 8). On
ultrathin sections from Euglena cells grown on hexanoic acid, antibodies against the MFP were significantly bound to mitochondria exclusively when gold
particle-conjugated secondary antibodies were used
for visualization (Fig. 7). No significant binding of the
conjugated gold particles was observed when using
preimmune serum in control experiments. Thus the
mitochondria are the only compartment in Euglena
containing the MFP.
Physiological Significance of the MFP
Adding 0.1 m cycloheximide to algae growing with
hexanoic acid stopped the increase of the l-3hydroxyacyl-CoA dehydrogenase, whereas chloramphenicol and rifampicin were not effective. These
results suggest that the MFP is neither coded nor
synthesized in mitochondria, but is translated in the
Plant Physiol. Vol. 131, 2003
cytoplasm. To find whether the increase of enzyme
activity was due to de novo synthesis or activation of
pre-existing enzymes, we compared the enzyme levels in hexanoic acid, oleic acid, and photoautotrophically grown algae by western-blot analysis. Crude
homogenates prepared from the same amount of algae were separated in Suc gradients. Fractions with
main activity for the dehydrogenase reaction were
separated in SDS gels, and proteins were electrotransferred to polyvinylidene difluoride membranes.
The blotted proteins were probed using anti-MFP
serum followed by anti-mouse IgG conjugated with
alkaline phosphatase. In all cases, only the three
bands of the MFP subunits appeared on the blots
(Fig. 8). The increases of enzyme levels after the
addition of hexanoic or oleic acid were immunochemically shown to be an increase in the amounts of
enzyme proteins (Fig. 8). These results demonstrate
that the MFP is induced by the short-chain saturated
fatty acid but also by the long-chain unsaturated fatty
acid, indicating that the Euglena enzyme complex is
capable of metabolizing a broad range of substrates.
DISCUSSION
Algae are capable of metabolizing fatty acids via
the ␤-oxidation pathway present in either mitochondria or microbodies or both of them (Stabenau, 1992).
But little is known about the enzymes participating
in this process. Especially lacking is information on
biochemical and structural properties of the algal
␤-oxidation enzymes. In Euglena, as demonstrated in
this paper, a tetrafunctional oligomeric protein is
involved in the metabolism of fatty acid-CoA esters
when the cells are grown on hexanoic or oleic acid.
Immunocytological experiments revealed that this
MFP is located in the mitochondria. The enzyme is
composed of the two smaller 32- and 34-kD subunits
and two larger subunits having molecular masses of
44.5 and 45.5 kD, respectively. The latter two subunits house the enzymes enoyl-CoA hydratase and
thiolase, whereas two different hydroxylacyl-CoA
dehydrogenases are present on the smaller subunits.
Epimerase activity, also measurable in the Euglena
MFP, could not be assigned to a special subunit,
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757
Winkler et al.
matrix enzymes in complexes would enable substrate
channeling like the binding of enzymes to membranes
and could also facilitate the tuning between the first
and the last reactions of the ␤-oxidation (Kunau et al.,
1995). Examples of these kinds of enzyme complexes
are the trifunctional protein isolated from rat liver
mitochondria (Uchida et al., 1992) and also the tetrafunctional MFP from Euglena mitochondria described
Figure 4. Distribution of proteins obtained by AS precipitation
(52.5%–62.5%) in a Suc gradient. A, Distribution of 3-hydroxyacylCoA dehydrogenase (Dehydrogenase) and 2-enoyl-CoA hydratase
(Hydratase) in the gradient. Enzyme activity is expressed as micromoles per minute per milliliter fraction. The distribution of molecular
masses for proteins separated in the gradient was obtained from the
distribution of standard proteins. B, Analysis of the proteins in the
different fractions of the gradient by SDS-PAGE and Coomassie
staining. Proteins were separated in 5% to 15% (w/v) gradient gels.
Lane M, Molecular mass markers as indicated on left. The arrows
indicate the proteins which correlate with the main peak of activity
for 3-hydroxyacyl-CoA dehydrogenase in fraction 11 of the gradient.
which may be due to the epimerization mechanism
(Hiltunen et al., 1989). The data that we obtained
indicate that acyl-CoA esters are degraded via
l-isomers of hydroxyacyl-CoA. Therefore epimerization of d-isomers to the l-form seems to be necessary
in MFPs, which are only capable of metabolizing the
l-forms of 3-hydroxyacyl-CoA esters as in Euglena.
The degradation of solely acyl-CoA esters via
l-isomers of substrates is a common characteristic of
mitochondrial ␤-oxidation systems. Long-chain acylCoAs undergo one or more cycles of chain shortening
catalyzed by long-chain-specific monofunctional enzymes that are bound to the inner mitochondrial
membrane (Carpenter et al., 1992). The binding to
membranes facilitates substrate channeling. It has
been speculated that the organization of participating
758
Figure 5. Enzyme purification on a dimethylaminoethyl anionexchange column. The proteins not bound to the sulfopropyl anionexchange column were injected on the column and eluted in a
gradient of 0 to 500 mM KCl prepared in 10 mM HEPES, pH 7.5,
containing 1 mM dithiothreitol (DTT) and 0.1% (v/v) Tween 20. The
KCl concentration increased by 10 mM per minute. Fractions of 1.5
mL were collected. A, Distribution of enzyme activities in the fractions eluted from the column. Enzyme activity is expressed as micromoles per minute per milliliter fraction for 3-hydroxyacyl-CoA
dehydrogenase (Dehydrogenase). Full-scale activity corresponds to
50 nmol min⫺1 mL⫺1 fraction for 2-enoyl-CoA hydratase (Hydratase), thiolase, and epimerase. B, Monitoring the purification by
PAGE. Lanes under I, 5% to 15% (w/v) SDS gradient gel of the
fractions eluted from the column. In fractions 4 to 6, only two 32and 34-kD bands are present. In fractions 7 to 9, an additional 45-kD
band appears. Lanes under II, 5% to 12% (w/v) SDS gradient gel from
fraction 7. Two separated bands are visible at 45 kD. Lanes under III,
Preparative native gel from fraction 7 containing only one single
protein. Lanes under IV, 5% to 15% (w/v) SDS gradient gel of the
protein extracted from the native protein in lane III: All three bands
that are present in the SDS gel from fraction 7 (Lanes under I) are
seen. Lane M, Molecular mass markers as indicated on the left. The
right arrows indicate the molecular masses of the subunits from the
purified protein.
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Plant Physiol. Vol. 131, 2003
Multifunctional ␤-Oxidation Enzyme in Euglena
Table II. Enzymatic and molecular properties of the MFP from Euglena
Enzymatic Properties
Molecular Mass
Optimum reaction pH
(K-phosphate buffer)
Km value
Ratio of enzyme functions
Dimethylaminoethyl
column
␮mol L
kD
3-Hydroxyacyl-CoA dehydrogenase
2-Enoyl-CoA hydratase
Thiolase
Epimerase
Native Protein
Subunits
a
Substrate was acetoacetyl CoA.
⫺1
50a
70b
6.5
6.25
Native gel
%
100
1.9
2.2
0.9
100
1.4
4.2
1.2
460
45.5, 44.5, 34, and 32
b
Substrate was crotonoyl CoA.
in this paper. It seems that both of the MFPs serve the
same purpose, although they are different with respect to their enzyme functions and their structural
organization. For example, only the Euglena enzyme
involves epimerase activity. Enoyl-CoA hydratase and
the 3-hydroxyacyl-CoA dehydrogenase are usually
domains of a common polypeptide. The Euglena enzyme is the only exception, because both of the enzyme functions are located on different subunits. The
Euglena MFP subunits are comparable with subunits
of monospecific enzymes with comparable functions.
For example, the subunits with 32 to 34 kD of l-3hydroxyacyl-CoA dehydrogenases from the archeon
Archeoglobus fulgidus (Klenk et al., 1997) and from
animal mitochondria (Osumi and Hashimoto, 1980)
correspond in size and function to the small subunits
of the Euglena MFP. The 43- to 45-kD subunits of the
2-enoyl-CoA hydratases from the archeon A. fulgidus
(Klenk et al., 1997) and from Closterium acetobutylicum
(Waterson et al., 1971) and the 42- to 45-kD subunits
for monospecific thiolases from various organisms
(Kunau et al., 1995) are comparable with respect to
molecular sizes and enzyme functions to the large
subunits of the Euglena MFP. Put together, these data
suggest that the Euglena MFP is a combination of
subunits from monospecific enzymes assembled in a
multienzyme complex.
The evolution of ␤-oxidation enzymes is characterized by two strategies for achieving the advantageous substrate channeling: integration of enzymes
into membranes and formation of multienzyme complexes. Only in the archeon A. fulgidus that is the
phylogenetically oldest organism known to possess
the ␤-oxidation pathway, all functions are distributed among separate enzymes (Klenk et al., 1997).
During evolution, multienzyme complexes were
formed by combining monospecific enzymes through
non-covalent interactions and/or gene fusion. The
MFP from Euglena is the first example of a multienzyme complex formed exclusively by non-covalent
interactions. The genome analysis of the archaeobacterium Pyrobaculum aerophilum (Fitz-Gibbon et al.,
2002) led to the discovery of the fusion of the genes
for 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA
dehydrogenases. The products of this gene, however,
are not known. The first common polypeptides having hydratase and dehydrogenase functions, probably forming a more stable gene product, occur in the
␣-subunits of multifunctional fatty acid oxidation
complexes of eubacteria like E. coli or Mycobacterium
leprae (Binstock et al., 1977; Cole et al., 2001). The
␣-subunits carrying the hydratase and dehydrogenase functions are combined by non-covalent interactions to a subunit housing a thiolase function in the
bacterial enzymes and in the trifunctional protein
from rat liver mitochondria (Uchida et al., 1992). The
peroxisomal and glyoxysomal MFPs only consist of
the product of the fused genes (Yang et al., 1991;
Kindl, 1993).
Although information on the amino acid sequence
of the Euglena MFP is limited to the N-terminal of the
32- and 34-kD subunits, significant alignments to
other ␤-oxidation enzymes were produced, allowing a
discussion on the role of Euglena MFP in the evolution
of multifunctional ␤-oxidation enzymes. Euglena is
believed to be one of the oldest known eukaryotic
organisms (Tessier et al., 1997). Therefore the Euglena
MFP should be more closely related to bacterial enzymes than to enzymes of higher developed eukaryotic organisms. In agreement with this assumption, it
Table III. Stereospecificity of the 2-enoyl-CoA dehydratase function of the MFP purified from Euglena
Enzyme Reaction
Substrate
Substrate Concentration
nmol mL
Hydratase
Dehydratase
Dehydratase
Plant Physiol. Vol. 131, 2003
Crotonoyl-CoA
S-3-Phenyl-2-propenoyl-CoA
R-3-Phenyl-2-propenoyl-CoA
⫺1
15
25
25
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Activity
nmol min⫺1 mL⫺1 fraction
85.0
0.4
15.4
759
Winkler et al.
Figure 6. Alignment of the amino-terminal sequences of 32- and 34-kD subunits of the Euglena MFP with several
␤-oxidation enzymes. Individual homologies were obtained with the BLAST program (see “Materials and Methods”). For the
32-kD subunit from Euglena, the identity with A. fulgidus is 51%, the similarity is 62%; the identity with Rattus sp.
peroxisomal MFP I is 51%, the similarity is 67%; the identity with P. aerophilum is 47%, the similarity is 65%; the identity
with M. musculus is 49%, the similarity is 59%; the identity with Caenorhabditis sp. is 38%, the similarity is 62%; the identity
with Arabidopsis is 46%, the similarity is 59%; the identity with Myobacterium sp. is 38%, the similarity is 54%; the identity
with Cucumis sp. is 31%, the similarity is 44%; the identity with Rattus sp. mitochondrial MFP is 31%, the similarity is 49%;
the identity with Euglena, 34-kD subunit is 51%, the similarity is 61%. For the 34-kD subunit from Euglena, the identity with
Acinetobacter sp. is 39%, the similarity is 50%; the identity with Pseudomonas sp. is 36%, the similarity is 47%. Amino acids
identical with the 32- and 34-kD subunits are shaded. Aligned sequences are indicated by the first amino acid. The GxGxxG
motive representing a conserved sequence around the NAD-binding side of dehydrogenases is boxed in. 3-HOADH,
3-Hydroxyacyl-CoA dehydrogenase; ECH, 2-enoyl-CoA hydratase; TFP, tetrafunctional protein. The numbers in parentheses
are the GenBank accession numbers from the National Center of Biotechnology Information.
was found that the 32-kD subunit of the Euglena MFP
shows a degree of similarity to the 3-hydroxyacyl-CoA
dehydrogenases from A. fulgidus. These data, plus the
similarities in the sizes of subunits between the Euglena MFP and the monospecific enzymes from A.
fulgidus, provide evidence that the monospecific enzymes from the archeon could possibly represent the
phylogenetic ancestors of Euglena MFP. In contrast to
the archeon, there was only a low degree of similarity
to the 3-hydroxyacyl-CoA dehydrogenases domain in
the multienzyme complex of fatty acid oxidation from
the eubacterium M. leprae, similar to the multifunctional fatty acid oxidation complex in E. coli. From the
analysis of the 16S rRNA, it is also established that
Figure 7. Thin sections of LR White resin embedded Euglena cells
grown on hexanoic acid. A, Sections were labeled by the IgG gold
method using antibodies against the Euglena MFP. The gold particles
representing the sites of the antigen are localized exclusively over the
mitochondria. B, Sections were labeled by the IgG gold method using
the preimmune serum of the immunization experiment. Bars ⫽ 0.1 ␮m.
760
Euglena is phylogenetically more related to archaebacteria than to eubacteria (Giovanni et al., 1988).
Therefore, one must consider that the Euglena enzyme
could possibly represent an additional phylogenetic
line of development for ␤-oxidation enzymes, different from the E. coli enzyme. This assumption would
imply a polyphyletic development of ␤-oxidation systems, as was also proposed for plastids (Bhattacharya,
1995). The significance of Euglena MFP in the evolu-
Figure 8. Immunoblot analysis of anti-MFP antibodies. Proteins of
crude homogenates were separated in Suc gradients. Each crude
homogenate contained the same amount of algal fresh weight peak
fractions with activity for 3-hydroxyacyl-CoA dehydrogenase were
electrophoresed on an SDS gel, and proteins were transferred to
blotting membranes. The membrane was incubated with anti-MFP
antibodies. The antibody-antigen reaction was detected by using
alkaline phosphatase. Lane M, Molecular mass marker. Lane A, MFP
from autotrophically grown algae. Lane C6, MFP from hexanoic
acid-grown algae. Lane C18:1, MFP from oleic acids-grown algae.
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Plant Physiol. Vol. 131, 2003
Multifunctional ␤-Oxidation Enzyme in Euglena
tion of ␤-oxidation enzymes, however, still has to
examined using more detailed sequence data.
dl-hydroxybutyryl-CoA (Binstock and Schulz, 1981). The protein concentration was measured using the protein assay (Bio-Rad, Hercules, CA;
modified Bradford method) according to the manufacturer’s instructions.
Bovine serum albumin was used as the standard.
MATERIALS AND METHODS
Algal Material and Growth Conditions
Gel Electrophoresis and Immunoblotting
Euglena (Euglena gracilis), strain 1224-5/25, was obtained from the collection of algal cultures at the University of Göttingen (Germany). The
nutrient medium contained 10 mm (NH4)2HPO4, 4 mm NaH2PO4, 5 mm
K2HPO4, 1 mm MgSO4, and 0.1 mm CaCl2. To 1 L of medium were added:
1 mg of B12, 1 mg of B6, 0.05 mg of B12, 1 mL of micronutrient solution, and
1 mL of Fe-EDTA complex (Kuhl and Lorenzen, 1964). The algae were
grown in glass tubes at 25°C under aeration with air plus 2% (v/v) CO2
either in continuous light of 40 ␮mol quanta m⫺2 s⫺1 or in the dark after the
addition of 5 mm hexanoic acid or oleic acid.
SDS-PAGE was carried out in gradient gels using standard methods (Laemmli, 1970). Native gel separations were performed as described by Laemmli
(1970) omitting SDS and 2-mercaptoethanol. For isoelectric focusing, the
method described by Robertson et al. (1987) was applied. A Bio-Rad mini
protean II cell was used for all separations. Gels were stained in 0.1% (w/v)
Coomassie Brilliant Blue R 250 in a mixture of 40% (v/v) methanol and 10%
(w/v) acetic acid and destained several times in a mixture of 40% (v/v)
methanol and 10% (w/v) acetic acid. Immunoblot analysis was performed
according to the method of Towbin et al. (1979). Antibodies were diluted
1:500 (v/v).
Cell Homogenization and Enzyme Purification
Algae (5 g fresh weight) were washed and homogenized in a grinding
buffer containing 10 mm HEPES, pH 7.5, 1 mm DTT, and 0.1 mm phenylmethylsulfonylfluoride. Cells were disrupted in a Virtis homogenizer in the
presence of glass beads (0.5 mm). The homogenate was filtered (nylon sieve,
0.2-mm pore size) and centrifuged at 1,100g for 5 min. The resulting supernatant was used for measuring the activities of the ␤-oxidation enzymes and
for protein separation in Suc gradients.
Molecular Mass Determination
The molecular masses of the proteins were determined after Suc gradient
centrifugation as described by Martin and Ames (1961). Catalase, malate
dehydrogenase, hexokinase, and peroxidase were used as Mr standards.
Production of Antibodies and
Immunoelectron Microscopy
qEnzyme Purification
The crude homogenate was incubated with 500 mm KCl at 5°C for 30 min,
followed by a treatment with 0.1% (v/v) Tween 20 for 30 min at 5°C. After
centrifugation at 48,000g for 30 min, proteins in the supernatant were
precipitated with 52.5% to 62.5% (of saturation) AS, collected by centrifugation, and suspended in 2 mL of grinding buffer containing 0.1% (v/v)
Tween 20. The AS precipitate was layered onto a Suc gradient from 5% to
20% (w/w) Suc prepared in 30 mL of the same buffer and centrifuged at
27,000 rpm (140,000g) in a SW 28-rotor (Beckman Coulter, Fullerton, CA) at
20°C for 18 h. Fractions of 2 mL were collected.
The following protein chromatography was performed on an inert MerckHitachi system using the following columns: a Fractogel EMD-sulfopropyl650 (S) cation-exchange column (Merck, Darmstadt, Germany) and a Fractogel EMD-dimethylaminoethyl-650 (S) anion-exchange column (Merck).
Fractions 10 to 12 from the Suc gradient were first applied to the cationexchange column and eluted in 10 mm HEPES, pH 7.5, containing 1 mm DTT
and 0.1% (v/v) Tween 20. The first 10 mL of the non-bound proteins were
collected and injected into the anion-exchange column. The bound proteins
eluted in a KCl gradient of 0 to 500 mm prepared in 10 mm HEPES, pH 7.5,
containing 1 mm DTT and 0.1% (v/v) Tween 20. The KCl concentration
increased by 10 mm per minute. Fractions of 1.5 mL were collected.
The MFP separated by Suc density centrifugation was separated from
contaminating proteins in a preparative native gel. The gel slice containing
the MFP was used as an antigen probe for the production of antibodies in
rabbits according to the protocol of the manufacturer (Bioscience, Germany).
The serum was partially purified on a cation-exchange column (Fractogel
EMD-SO3-650 (S), Merck) according to the manufacturer’s instructions. For
electron-microscopic preparation, the algae were fixed with 1.0% (v/v)
glutaraldehyde and 4% (v/v) depolymerized paraformaldehyde in a 100
mm cacodylate buffer, pH 7.4, at 20°C for 2 h. Cells were dehydrated in a
graded ethanol series. Embedding in LR White resin was performed with a
mixture of 33% and 66% (w/v) LR White resin in ethanol followed by three
incubations with pure resin. All steps were carried out at 5°C for 6 h. Blocks
were polymerized in gelatin capsules at 50°C for 30 h. Ultra-thin sections
were mounted on uncoated nickel grids. The following steps were all
performed at 30°C: The sections were treated with a blocking solution (2.5%
[w/v] nonfat dry milk in phosphate buffered saline) for 30 min and then
incubated for 2 h in a solution of the antibody diluted 1:100 (v/v) in
blocking medium. After washing with phosphate buffered saline, the sections were incubated with goat anti-rabbit IgG conjugated to 15-nm gold
particles (British Biocell, Cardiff, UK), diluted 1:20 (v/v) in a blocking
medium for 1 h. The sections were washed with distilled water, and stained
with 4% (w/v) uranyl acetate and lead citrate.
Assays
Enoyl-CoA hydratase activities were measured as the hydration of
crotonoyl-CoA with 15 ␮m crotonoyl-CoA as the substrate (Steinmann and
Hill, 1975). The reverse reaction, dehydration of 3-hydroxyacyl-CoA esters,
was observed by measuring the increase of A308 due to the formation of
3-phenyl-2-propenoyl-CoA in an assay containing enzyme fraction, 200 mm
phosphate buffer, pH 8.0, 25 ␮m of the R- or S-isomers of 3-hydroxy-3phenylpropionyl-CoA and 1 m EDTA (Baes et al., 2000). The activity of
3-hydroxyacyl-CoA dehydrogenase was monitored by using the oxidation of
NADH in the presence of 15 ␮m acetoacetyl-CoA (Overath and Raufuss,
1967). Thiolase activity was observed by measuring the dissociation of the
Mg-acetoacetyl-CoA complex at 303 nm (Middleton, 1975) using 15 ␮m
acetoacetyl-CoA.
Epimerase activity was monitored by reducing NAD⫹ after complete
oxidation of the l-isomer of dl-hydroxybutyryl-CoA by adding l-3hydroxyacyl-CoA dehydrogenase (bovine liver) in a test medium 30 ␮m
Plant Physiol. Vol. 131, 2003
Protein Microsequencing and Search for Homologies
Protein N-terminal sequencing was performed by automated Edman degradation with electrophoretically purified enzyme subunits blotted on polyvinylidene difluoride membranes. Sequence homologies were determined
using the BLAST program (Altschul et al., 1997) of the National Center for
Biotechnology.
ACKNOWLEDGMENTS
We thank Dr. F. Buck (Institut für Zellbiologie und Klinische Neurobiologie, Universtität Hamburg) for the analysis of the amino acid sequences.
Received August 19, 2002; returned for revision October 1, 2002; accepted
October 28, 2002.
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761
Winkler et al.
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