(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 -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.
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
(-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) .
Figure 1:
Reciprocal stereochemical
pathways for the -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).
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 -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.
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 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.
Single colonies from YPD or ura 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.
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 -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.