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(Received for publication, July
17, 1995; and in revised form, September 12, 1995) From the
The biosphere is inherently built of chiral molecules, and once
their metabolism is established, the stereochemical course of the
reactions involved is seen to remain highly conserved. However, by
replacing the yeast peroxisomal multifunctional enzyme (MFE), which
catalyzes the second and third reactions of
Fatty acids are universal constituents of living cells, and they
are used as essential components of biomembranes and as a store for
combustion energy(1) . Their degradative pathway
(
Figure 1:
Reciprocal stereochemical
pathways for the
Using yeast cells carrying a
deletion of the MFE gene (YFOX-2)(18) , we studied whether the
stereochemistry plays a role in the evolution of
Figure 2:
Construction of expression plasmid
carrying rat peroxisomal MFE behind yeast oleic acid-inducible catalase
A promoter. pYE352-CTA1 was constructed from YEp352-CTA1 by adding a
linker sequence containing an extra XhoI restriction site (A), and the pYE352-rMFE was created by joining the rat MFE
cDNA behind the yeast catalase promoter by digesting pYE352-CTA1 and
pUC18-rMFE with SacI and XhoI and ligating them
together (B). PCR, polymerase chain
reaction.
The
coding region of cDNA for rat MFE (25) was obtained from total
RNA isolated from clofibrate-treated rat liver by reverse transcription
with moloney murine leukemia virus reverse transcriptase and
amplification by polymerase chain reaction using rat MFE-specific
primers and cloned into pUC18 vector using the Sure Clone cloning kit
(Pharmacia, Uppsala, Sweden) This pUC18-rMFE was used as template for
polymerase chain reaction with specific oligonucleotide primers
containing unique restriction sites in their 5` ends. The resulting
rMFE cDNA had an extra SacI restriction site in the 5` end and
an XhoI cutting site in the 3` end and was cloned in pUC18
(pUC18-rMFE(SacXho)) in order to allow the amplification of the
fragment (Fig. 2B). pYE352-CTA1 and
pUC18-rMFE(SacXho) were digested with SacI and XhoI,
and the fragments containing the rat MFE and pYE352 with catalase
promoter were isolated from 0.7% agarose gel by Geneclean II kit (Bio
101 Inc., Vista, CA) and ligated overnight. The resulting construct
contains the rat MFE open reading frame under the control of oleic
acid-inducible catalase promoter, and the YFOX-2 cells were transformed
by lithium acetate method(26) . The transformants were selected
on ura
Single colonies from YPD or ura
For measuring the L-specific 3-hydroxyacyl-CoA dehydrogenase activity, 60
µmol of a racemic mixture of D,L-3-hydroxydecanoyl-CoA was incubated in the
Tris/HCL/bovine serum albumin/potassium chloride mixture (see above)
containing 25 µM magnesium chloride, 1 µM sodium pyruvate, 10 µg of lactate dehydrogenase from rabbit
muscle (Boehringer Mannheim) in the presence of recombinant D-specific 3-hydroxyacyl-CoA dehydrogenase. After the D-isoform was removed, the reaction was initiated by adding
the sample. When assaying the D-specific 3-hydroxyacyl-CoA
dehydrogenase, the L-isoform of the substrate was first
removed from the reaction mixture with the L-specific
3-hydroxyacyl-CoA dehydrogenase followed by adding the sample. The
oxidation of 3-hydroxydecanoyl-CoA was followed by monitoring the
formation of magnesium-3-ketodecanoyl complex at 303
nm(18, 28, 29) . Recombinant D-specific 3-hydroxyacyl-CoA dehydrogenase was a truncated
version of the peroxisomal MFE from Candida tropicalis, and it
was produced as described by Hiltunen et al.(18) .
Stereospecificities of L- and D-hydroxyacyl-CoA
dehydrogenases have been tested separately using synthetized L- and D-isoforms of 3-hydroxydecanoyl-CoA, which
were obtained as described by Malila et al.(29) .
Figure 3:
Complementation of fox-2 mutant
of S. cerevisiae by rat peroxisomal MFE. The wild-type strain
UTL-7A (1), fox-2 mutant devoid of yeast peroxisomal
MFE (2), fox-2 mutant transformed with pYE352-CTA1
encoding catalase A (3), and fox-2 mutant strain
transformed with pYE352-rMFE containing the cDNA of rat peroxisomal MFE
under the control of catalase A promoter (4). The strains were
grown on rich medium, YPD (A), and on a medium containing 0.1%
oleic acid as a carbon source (B).
Figure 4:
Northern and Western blot analyses of
the yeast strains used cultivated in different media. A,
Northern blot. Lane 1, UTL-7A grown in YPD; lane 2,
UTL-7A grown in oleic acid; lane 3, YFOX-2-rMFE grown in
SC-ura; lane 4, FOX-2-rMFE grown in oleic acid medium. The
sizes of yeast rRNAs are given on the left. B,
Western blot analysis. The strains, media, and sample order are as in A. The purified wild-type rat peroxisomal MFE was used as
sample in lane 5.
In
immununoblot analysis of soluble proteins from the four yeast strains
using antibodies against rat MFE, a signal of 78 kDa was obtained only
with proteins from YFOX-2-rMFE cells grown in oleic acid medium (Fig. 4B), which corresponds well with the signal
obtained with the rat wild-type MFE purified from rat
liver(22) .
Figure 5:
Stereospecificity of 3-hydroxyacyl-CoA
dehydrogenase from wild-type and transformed yeast cells. A shows the experiment carried out with YFOX-2-rMFE cells grown on
oleic acid. B shows the experiment carried out with UTL-7A
cells grown on oleic acid. The labeled arrows indicate the
time of addition of 90 µg of soluble extract from UTL-7A (1), 10 µg of L-3-hydroxyacyl-CoA dehydrogenase (2), 2 µg of D-3-hydroxyacyl-CoA dehydrogenase (3), and 90 µg of soluble extract from YFOX-2-rMFE (4). In the assay 20 nMD,L-3-hydroxydecenoyl-CoA was used as
substrate.
When soluble proteins were extracted from UTL-7A and
YFOX-2-rMFE yeast strains grown on glucose and oleic acid media and the
samples were tested for combined activity (i.e. capability to
convert trans-2-decenoyl-CoA to 3-ketoacyl-CoA), activity was
observed within the detection limits only in oleic acid-grown cells (Table 2).
When enzyme activities participating in
3-hydroxyacyl-CoA metabolism were studied in more detail, activities of
2-enoyl-CoA hydratase 2 and D-3-hydroxyacyl-CoA dehydrogenase
could be detected in extracts of UTL-7A cells grown on oleic acid (Table 2). Similarly, the combined activity (rate of metabolism
of trans-2-decenoyl-CoA to 3-ketoacyl-CoA) was also present.
Furthermore, in UTL-7A extracts the activities of 2-enoyl-CoA hydratase
1 and L-3-hydroxyacyl-CoA dehydrogenase were below the
detection limits in the assay system used. The extracts from
YFOX-2-rMFE cells contained both 2-enoyl-CoA hydratase 1 and L-3-hydroxyacyl-CoA dehydrogenase activities but were lacking D-3-hydroxyacyl-CoA dehydrogenase activity. Unexpectedly,
about 42% of hydratase 2 activity was still detectable when compared
with the wild-type cells. Because it has been shown earlier that the
mRNA for FOX-2p (yeast peroxisomal MFE) is not produced in YFOX-2
cells, this result indicates that in addition to FOX-2p there exists
another protein capable of catalyzing hydratase 2 reactions in yeast.
Interestingly, recent data have shown that in rat liver hydratase 2
activity can be found in two subcellular compartments: peroxisomes and
microsomes(29) .
Figure 6:
Immunoelectron microscopy of cultured
yeast cells labeled with antibodies to rat peroxisomal MFE. Wild-type
cells (UTL-7A) (A) and FOX-2-rMFE cells (B) cultured
in oleic acid medium. Bars, 100
nm.
Present work provides several lines of evidence that the rat
peroxisomal MFE can be heterologously expressed in an active form in S. cerevisiae and that it complements the lack of the
corresponding endogenous yeast peroxisomal MFE. (i) Both the mRNA and
the rat protein were detected only in cells transformed with plasmid
pYE352-rMFE. (ii) The transformants had gained 2-enoyl-CoA hydratase 1
and L-3-hydroxyacyl-CoA dehydrogenase activities, which were
undetectable in wild-type yeast cells. (iii) Immunoelectron microscopy
indicated that the rat MFE was targeted into the peroxisomes. (iv)
Finally, the expression of rat peroxisomal MFE allows the YFOX-2 mutant
cells to utilize oleic acid as a carbon source. The observed
functional complementation, however, results in the change of
stereochemistry of the second (hydration) and the third (oxidation)
reactions of
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27453-27457
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EXPERIMENTS WITH THE PEROXISOMAL 
-OXIDATION IN YEAST (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-oxidation of fatty
acids via D-3-hydroxyacyl-CoA intermediates, with rat
peroxisomal MFE, which catalyzes the same reactions via L-3-hydroxy intermediates, it was possible to change the
chiralities of the intermediates in a major metabolic pathway in
vivo. Both stereochemical alternatives allowed the yeast cells to
grow on oleic acid, implying that when the -oxidation pathways
evolved, the overall function was the determining factor for the
acquisition of MFEs and not the stereospecificities of the reactions
themselves.
-oxidation) was found to be confined to peroxisomes in all
eukaryotic cells (2, 3, 4) and additionally
to mitochondria in animal cells(5, 6) . Until
recently, the second and third reactions of -oxidation, which are
catalyzed by multifunctional enzymes (MFEs) (
)in
extramitochondrial systems (7, 8, 9) and with
very long-chain substrates also in mitochondria(10) , were
assumed to proceed in all organisms via L-3-hydroxyacyl-CoA
esters when degrading fatty acids (11, 12, 13) and via D-3-hydroxy-intermediates in the de novo synthesis of
fatty acids(14, 15, 16, 17) .
However, the observation that -oxidation in the peroxisomes of Saccharomyces cerevisiae occurs via D-3-hydroxy
metabolites (18) opened up a new perspective on the evolution
of -oxidation systems (Fig. 1). The peroxisomal MFEs of
higher (mammals and plants) and lower eukaryotes (fungi) not only
catalyze two reactions of opposite chiral specificity, but they also
have different native molecular sizes and distinct amino acid
sequences, indicating different phylogenetic
origins(18, 19) .
-oxidation of fatty acids. The activity
catalyzing the hydration of trans-2-enoyl CoA to L-3-hydroxyacyl intermediates is called here 2-enoyl-CoA
hydratase 1 (EC 4.2.1.17) (Hydratase 1), and that catalyzing
the hydration of trans-2-enoyl CoA to D-3-hydroxyacyl
intermediates is called 2-enoyl-CoA hydratase 2 (EC 4.2.1.-) (Hydratase 2). The dehydrogenation reactions are as follows: L-specific 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) (L-HADH) and D-specific 3-hydroxyacyl-CoA hydrogenase
(EC 1.1.1.-) (D-HADH).
-oxidation
systems. As fungi possess only a peroxisomal
-oxidation(20, 21) , this yeast mutant does not
degrade fatty acids and is thus unable to grow on fatty acids as a
carbon source. We replaced the yeast endogenous MFE, comprising
2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA
dehydrogenase activities(18) , with its peroxisomal counterpart
in the rat, possessing enoyl-CoA hydratase 1 and L-specific
3-hydroxyacyl-CoA dehydrogenase activities(7, 22) .
This replacement resulted in functional complementation giving the
YFOX-2 cells back their capability to grow on oleic acid.
Strains and Media
The S. cerevisiae strains and plasmids used are listed in Table 1. Escherichia coli strain DH5
was used for all
transformations and plasmid isolations.
Plasmid Construction
Into a YEp352 vector
containing the catalase A sequence from S. cerevisiae (CTA1) (23) and modified as described by Kragler et
al.(24) , an additional unique restriction site was
introduced by cutting with HindIII, dephosphorylating, and
ligating again in the presence of a polynucleotide kinase-treated
oligonucleotide representing the XhoI site
(5`-AGCTTCTCGAGA-3`). The resulting plasmid pYE352-CTA1 contains the
whole CTA1 gene with modifications allowing the replacement of the open
reading frame by cutting with XbaI or SacI and HindIII or XhoI (Fig. 2A).
plates and replica plated on oleic acid
plates. Colonies growing on both plates were chosen for complementation
studies. UTL-7A, YFOX-2, and YFOX-2-CTA1 strains were used as controls.
Growth Conditions and Harvesting the Cells
Yeast
cells were cultured at 30 °C in the following media: 1% yeast
extract, 2% Bacto-peptone, 2% glucose (YPD); 2% glucose, 0.67%
Bacto-yeast nitrogen base without amino acids, 0.2% dropout powder
without uracil (27) (SC-ura); 0.1% oleate, 0.5% yeast extract,
0.5% Bacto-peptone, 0.1% glucose, 0.5% Tween 40, 6.4 ml of vitamin
solution/liter, 60 ml of salt solution/liter, pH 6.0 (oleic acid medium
with uracil); and 0.1% glucose, 0.1% oleic acid, 0.5% Tween 40, 0.3%
Bacto-yeast nitrogen base without amino acids, 0.2% dropout mix without
uracil, 60 ml of salt solution/liter, 6.4 ml of vitamin solution/liter,
pH 6.0 (oleic acid medium without uracil). Salt solution (as expressed
per liter) was as follows: 125 g of
(NH
)
SO, 25 g of
KHPO, 12.5 g of MgSO
7
HO, 2.5 g of NaCl, 2.5 g of CaCl, 10 mg of
ZnSOHO, 1 mg of CuSO5
HO, 10 mg of MnSOHO, 2.5 mg
of KI, 8.3 mg of FeCl
6 HO, 12.5 mg of
HBo, 5.9 mg of
NaMoOHO. Vitamin solution
(per liter) was as follows: 200 mg of thiamine, 200 mg of nicotinic
acid, 200 mg of calcium pantothenate, 200 mg of pyridoxine
hydrochloride, 100 mg of p-aminobenzoic acid, 100 mg of
riboflavin, 1 mg of biotin, 1 mg of folic acid, 1 g of myo-inositol. plates were grown overnight in YPD (UTL-7A) and in SC-ura
(YFOX-2-rMFE). 1-liter batches of YPD medium (UTL-7A), SC-ura
(YFOX-2-rMFE), oleic acid medium with uracil (UTL-7A), and oleic acid
medium without uracil (YFOX-2-rMFE) were inoculated with overnight
cultures to cell density of 1
10
cells/ml and grown
at 30 °C with shaking for 48 h. The cells were harvested by
centrifuging (2000 g for 10 min), washed twice with
sterile distilled water, and centrifuged as above.Northern Blot Analysis
The strains were grown in
liquid cultures, and the REX total RNA isolation kit from USB
(Cleveland, OH) was used for isolating total RNA from 0.5 g of yeast
cells from each culture. 10-µg samples of each RNA were
fractionated by electrophoresis in formaldehyde-agarose (0.8%) and
blotted onto a nitrocellulose filter. The filter was hybridized with
random prime labeled rat MFE probe.Immunoblot Analysis
For immunoblot analysis the
harvested cells were broken with glass beads, and the lysates were
subjected to SDS-polyacrylamide gel electrophoresis followed by
electroblotting on a nitrocellulose filter. The rat MFE protein was
immunodetected with rabbit anti-rMFE and using affinity-purified goat
anti-rabbit IgG with horseradish peroxidase conjugate (Bio-Rad) as
second antibody.Enzyme Activity Assays and Studies on Stereospecificities
of 2-Enoyl-CoA Hydratases and 3-Hydroxyacyl-CoA Dehydrogenases from
Wild-type and Transformed Yeast Cells
2-Enoyl-CoA hydratase 1
activity was measured in the direction of hydration of trans-2-decenoyl-CoA to L-3-hydroxydecanoyl-CoA. The
incubation mixture consisted of 60 nmol of trans-2-decenoyl-CoA in 50 µM Tris/HCl, pH 9.0,
50 µg of bovine serum albumin, 50 µmol of potassium chloride, 1
µmol of NAD, 5 µg of porcine L-3-hydroxyacyl-CoA dehydrogenase (Sigma) in a volume of 1 ml
at 23 °C. The reaction was started by adding a sample into the
cuvette and monitored by following the NADH production
spectrophotometrically. 2-Enoyl-CoA hydratase 2 activity was measured
in the direction of hydration of trans-2-decenoyl-CoA to D-3-hydroxydecanoyl-CoA under the conditions described above
except that the L-3-hydroxyacyl-CoA dehydrogenase was replaced
with 0.5 µmol
min of D-3-hydroxyacyl-CoA dehydrogenase activity (determined with D-3-hydroxydecanoyl-CoA).
Immunoelectron Microscopy
UTL-7A and FOX-2-rMFE
cells were grown on oleic acid, and the pelleted cells were fixed in 8%
paraformaldehyde, processed, and immunolabeled as described by Sormunen et al.(30) . The rabbit anti-rMFE IgG bound on cell
sections was localized with protein A-colloid gold labeling technique.
Growth of Yeast Cells on Different Media
All
yeast strains tested (UTL-7A, YFOX-2, YFOX-2-CTA1, and YFOX-2-rMFE)
were viable (Fig. 3) on glucose-rich YPD plates, whereas on
oleic acid plates only YFOX-2-rMFE and wild-type UTL-7A cells were able
to grow. In a control experiment on ura plates, only
the strains transformed with pYE352-rMFE or pYE352-CTA1 were viable
(data not shown). Altogether, these results indicate that apparent
complementation of fox-2 mutation by the rat MFE encoded by
the plasmid pYE352-rMFE had occurred.
Northern Blot and Immunoblot Analyses
To
investigate further the expression of rat MFE in YFOX-2-rMFE, Northern
blot analysis was carried out. The rat MFE cDNA probe hybridized only
with the RNA isolated from YFOX-2-rMFE cells grown in oleic acid
medium, the size of the signal (2.2 kb) corresponding well with the
estimated size of the rat MFE RNA (25) (Fig. 4A). This signal was not seen in lane 3 of Fig. 4A (YFOX-2-rMFE grown in
glucose medium), which shows that the CTA1 promoter is a strong
glucose-repressible and oleic acid-inducible promoter, which is in
agreement with earlier observations(31, 32) .
Enzyme Activity Assays
The yeast MFE and the rat
peroxisomal MFE are known to catalyze the conversion of trans-2-enoyl-CoA to 3-ketoacyl-CoA via hydration and
dehydrogenation steps, but the stereochemical courses of the reactions
are different; in yeast the events occur via D-3-hydroxyacyl-CoA, whereas in rat they occur via the
corresponding L-form(18, 28) . This
difference was used to design kinetic experiments to distinguish
between the two enzymes. As shown in Fig. 5A, when the
protein sample from oleic acid-grown UTL-7A cells was added to a
racemic mixture of the D- and L-forms of
3-hydroxydecanoyl-CoA, only one half of the substrate was converted to
3-ketodecanoyl-CoA. The subsequent addition of L-specific
3-hydroxyacyl-CoA dehydrogenase from porcine heart resulted in
consumption of the second half of the substrate. This indicated that
the yeast extract used contained only D-specific
hydroxyacyl-CoA dehydrogenase. This conclusion was further confirmed by
the observation that the addition of the recombinant D-3-hydroxyacyl-CoA dehydrogenase (18) did not result
in any additional production of 3-ketoacyl-CoA ester. When the
corresponding experiment was carried out with YFOX-2-rMFE rather than
wild-type cells, the addition of the cell lysate again caused the
removal of only half of the substrate (Fig. 5B), but
this time separate additions of the auxiliary enzymes, L-specific and D-specific hydroxyacyl-CoA
dehydrogenases, yielded opposite effects; the L-specific
enzyme caused no reaction, whereas the D-specific one
metabolized the remaining half of the substrate. These results clearly
demonstrate that the transformed YFOX-2 strain was devoid of its
endogenous D-3-hydroxyacyl-CoA dehydrogenase activity but
possessed the expressed L-3-hydroxyacyl-CoA dehydrogenase of
plasmid origin.
Subcellular Localization
To study the subcellular
location of rat MFE expressed in YFOX-2 cells, immunoelectron
microscopy was carried out applying protein A colloid gold technique
and using anti-rMFE antibodies. As shown in Fig. 6, most of the
gold particles were found within peroxisomes in YFOX-2-rMFE cells grown
on oleic acid in contrast to wild-type cells, for which no labeling was
found with the same procedure. This implies that the targeting signal
for rMFE is recognized by the yeast import mechanism.
-oxidation (Fig. 1), namely from D-3-hydroxyacyl-CoA-dependent pathway to L-3-hydroxy
intermediate-dependent one. Consequently, the results indicate that the
functioning of the -oxidation pathway in S. cerevisiae is
independent of the stereochemistry of its second and third reactions.
This gives rise to the question of why two chirally different
-oxidation pathways are found in higher and lower eukaryotes. The
simplest explanation, that both were present in a phylogenetic
ancestor, is contradicted by the fact that only one type of MFE has
been found in eubacteria(33, 34, 35) and
mammalian mitochondria (36) belonging to the same protein
family as those of animal (25, 37) and plant
peroxisomes(38) . It is more likely that this original MFE was
replaced by a new one (hydratase 1/L-specific 3-hydoxyacyl-CoA
dehydrogenase by hydratase 2/D-specific 3-hydroxyacyl-CoA
dehydrogenase) in the fungi after the bifurcation of animals and fungi.
Thus the results suggest that the acquisition of enzymes for a
metabolic pathway was based on the executing of a certain function and
not on the stereochemical course of the events. Following this
principle, the evolution of -oxidation pathways has resulted in a
special type of functional convergence in that the MFEs from yeast and
mammalian peroxisomes catalyze the same two sequential reactions but
via reciprocal stereochemistry.-Oxidation systems are usually
visualized as forming an organized structure in order to provide
efficient flux through the many sequential reactions of their
pathways(39, 40) . It therefore seems surprising at
first sight that a phylogenetically unrelated heterologous protein can
efficiently replace one endogenous component of this metabolon. It
cannot be excluded, however, that the two multifunctional proteins of
rat and fungal peroxisomes may be related in terms of their
three-dimensional structure. If this turns out to be true in the
future, it will mean that in addition to functional convergence,
structural convergence must also have occurred.
)
We thank A. Holappa, R. Pietilä,
I. Vuoti, and U. Dorpmund for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Gurvitz, B. Hamilton, H. Ruis, and A. Hartig Peroxisomal Degradation of trans-Unsaturated Fatty Acids in the Yeast Saccharomyces cerevisiae J. Biol. Chem., January 5, 2001; 276(2): 895 - 903. [Abstract] [Full Text] [PDF]
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