From the Department of Chemistry, City College and
Graduate School of the City University of New York, New York, New York
10031 and the ¶ Department of Pharmacology, New York State
Institute for Basic Research in Developmental Disabilities, Staten
Island, New York 10314
Received for publication, December 15, 2000
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ABSTRACT |
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The degradation of unsaturated fatty acids by Dienoyl-CoA isomerase was first identified in rat liver mitochondria
but later was also detected in rat liver peroxisomes (4, 5). The
molecular characterization of this enzyme revealed the amino acid
sequence of the unprocessed subunit, which has a peroxisomal targeting
signal, type 1, at the C terminus and an N-terminal sequence
that is consistent with the targeting of this protein to mitochondria
(6). This situation is suggestive of a dual subcellular localization
and agrees with the previously observed kinetic and immunological
similarities of the mitochondrial and peroxisomal forms of this enzyme
(5). The crystal structure of a recombinant form of dienoyl-CoA
isomerase consisting of subunits without the 53 N-terminal amino acid
residues was obtained at 1.5-Å resolution (7). This study confirmed
the proposed hexameric structure of the enzyme (6) and revealed the
active site as a deeply buried hydrophobic pocket with three acidic
residues, Asp176, Glu196, and
Asp204. The latter two of these residues were predicted to
catalyze proton transfers at carbons 2 and 6, respectively, of the
3,5-dienoyl-CoA substrate (6). Surprisingly,
The association of dienoyl-CoA isomerase and trienoyl-CoA isomerase
with the same protein prompted this study aimed at elucidating the
mechanisms of action of these enzymes. This goal necessitated the
characterization of the highly active, mature forms of these enzymes
present in mitochondria and peroxisomes.
Materials--
A PCR Advantage kit and a Transformer
site-directed mutagenesis kit were purchased from
CLONTECH. PGEM-T Easy was obtained from Promega.
Oligonucleotides were synthesized by Integrated DNA Technologies, Inc.
Restriction endonucleases, T4 polynucleotide kinase, T4 ligase, and T4
DNA polymerase were supplied by New England BioLabs. Nycodenz, CoASH,
NADPH, Polybuffer Exchanger 94, cyanogen bromide-activated Sepharose,
and most standard biochemicals were purchased from Sigma Chemical Co.
Hydroxylapatite, the dye reagent for protein assays, Sequi-Blot
polyvinylidene difluoride membrane, 4-20% polyacrylamide ready gels,
and the materials for immunoblotting, including the goat anti-rabbit
IgG conjugated with alkaline phosphatase, were bought from Bio-Rad.
Rabbit antiserum against dienoyl-CoA isomerase was raised by Pocono
Rabbit Farms and Laboratory, Canadensis, PA. Monospecific antibodies to
dienoyl-CoA isomerase were prepared from antiserum by affinity
chromatography using a Sepharose-dienoyl-CoA isomerase column prepared
from recombinant dienoyl-CoA isomerase and cyanogen bromide-activated
Sepharose. Male Harlan Sprague-Dawley rats from Taconic Farms,
Germantown, NY were fed rodent chow containing 2% (w/w)
di(ethylhexyl)phthalate for at least 2 weeks before use.
3,5-cis-Octadienoyl-CoA and
2,5-cis-octadienoyl-CoA were prepared as described by
Shoukry et al. (9). 3,5,7-Decatrienoyl-CoA was prepared as
described by Liang et al. (8). Contaminating 2,5-cis-octadienoyl-CoA and 2,5,7-decatrienoyl-CoA in
preparations of 3,5-cis-octadienoyl-CoA and
3,5,7-decatrienoyl-CoA, respectively, were removed by HPLC.
Preparations of 2,5-cis-octadienoyl-CoA and
2,5,7-decatrienoyl-CoA were purified by HPLC after converting residual
amounts of 3,5-cis-octadienoyl-CoA and
3,5,7-decatrienoyl-CoA to their 2,4 and 2,4,6 isomers, respectively, in
the presence of dienoyl-CoA/trienoyl-CoA isomerase.
Enzyme and Protein Assays--
All isomerases were assayed
spectrophotometrically with 20 µM substrate in 0.2 M potassium phosphate at pH 8. The isomerase activities
that were measured are indicated hereafter together with the
substrates, wavelengths, and extinction coefficients of the assays:
Purification of Rat Mitochondrial and Peroxisomal Dienoyl-CoA
Isomerases--
Dienoyl-CoA isomerases from rat liver and heart were
purified as described previously (3). Adult Harlan Sprague-Dawley rats
were used, which had been fed rodent chow containing 2% (w/w) di(ethylhexyl)phthalate. For the purification of rat liver peroxisomal dienoyl-CoA isomerase, a light mitochondrial fraction was prepared from
two rat livers as described by de Duve et al. (11).
Peroxisomes were prepared by Nycodenz density gradient centrifugation
of a light mitochondrial fraction as described previously (5). For this
purpose, a 30% (w/v) solution of Nycodenz containing 1 mM EDTA, 5 mM Hepes (pH 7.3), and 0.1% ethanol was prepared,
and 21 ml of this solution was placed in a 30-ml ultracentrifuge tube on top of 1.5 ml of a 60% sucrose cushion. A density gradient was
generated by centrifugation at 60,000 × g in a T865
small angle rotor on a DuPont RC70 ultracentrifuge at 4 °C for
24 h. A light mitochondrial fraction (~45 mg of protein in 1.5 ml) was layered on top of the gradient followed by 1.5 ml of a cover
solution of a 3-fold diluted isolation buffer containing 0.25 M sucrose, 1 mM EDTA, 0.1% ethanol, and 10 mM Tris-HCl (pH 7.4). The sample was centrifuged at
76,000 × g for 1 h at 4 °C. Fractions of 2.5 ml each were collected from the bottom of the tube. Peroxisomes, microsomes, and mitochondria were localized by assaying the marker enzymes catalase, esterase, and malate dehydrogenase, respectively. Peroxisomal fractions were combined and diluted 5-fold with isolation buffer before they were harvested by centrifugation at 17,500 × g for 20 min. Pellets were suspended in 2 ml of 5 mM potassium Pi (pH 6.3) containing 5 mM mercaptoethanol, 1 mM EDTA, 1 mM
EGTA, 1 mM benzamidine, and 0.5 mM PMSF (buffer
A). The suspension was centrifuged at 100,000 × g for
1 h after sonicating it 10 times for 20 s each at 4 °C.
The supernatant was applied to a hydroxylapaptite column (1.5 × 22 cm) previously equilibrated with buffer A. The column was washed
with buffer A containing 0.5 M KCl and then was developed
with a gradient made up of 160 ml of buffer A and 160 ml of buffer A
containing 0.8 M potassium Pi (pH 6.3).
Fractions of 3 ml each were collected, and the fractions containing the dienoyl-CoA isomerase activity were combined and concentrated in an
Amicon concentrator with a YM-10 membrane. After dialysis overnight
against 25 mM ethanolamine-acetic acid (pH 9.4)
containing 5 mM mercaptoethanol, 1 mM EDTA, 1 mM benzamidine, 0.5 mM PMSF, and 20% glycerol
(buffer B), the sample was applied to a chromatofocusing column (1 × 15 cm) containing Polybuffer Exchanger 94 equilibrated with buffer
B. The column was extensively washed with buffer B and then developed
with 12 column volumes of Polybuffer 96 adjusted to pH 6.0 with acetic
acid. Fractions of 3 ml each were collected and assayed for dienoyl-CoA
isomerase. The active fractions were combined and concentrated with a
Millipore centrifugal filter device.
SDS-PAGE and Immunoblotting--
Aliquots of purified
dienoyl-CoA isomerase were treated with SDS sample buffer and subjected
to SDS-PAGE on gradient (4-20%) gels (12). Proteins were transferred
to a polyvinylidene difluoride membrane by semi-dry blotting (13), and
proteins remaining on the gel were visualized by staining with
Coomassie Blue. The membrane was incubated for 1 h with a
500-fold-diluted rabbit antiserum or with monospecific antibodies (1 µg/ml) prepared from the serum raised against rat liver dienoyl-CoA
isomerase. After incubating the membrane with goat anti-rabbit IgG
conjugated with alkaline phosphatase, it was developed with a
staining mixture containing the alkaline phosphatase substrate until
the antigen bands were visualized (14).
Analysis of Protein Sequence--
N-terminal amino acid
sequencing was performed by Stephen Bobin at the Dartmouth College
Molecular Biology Core Facility. The N-terminal sequence of the
full-length dienoyl-CoA isomerase was analyzed with the program
HelicalWheel to draw a helical wheel as described previously
(15).
Cloning and Expression of Dienoyl-CoA Isomerase--
Rat
heart Marathon-Ready cDNA (CLONTECH) was used
as the template for cloning the cDNA of the full-length dienoyl-CoA
isomerase by touch-down PCR according to the protocol of
CLONTECH. The primers were
5'-CAGGATCCCATATGGCTACCGCGATGACAGTTTCCA-3' and
5'-CAGTAAGCTTATCAGAGCTTGGAGAAGGTGATGCTT-3'. The PCR product (~1 kb)
was inserted into vector pGEM-T Easy (Promega) and amplified.
Thereafter, it was subcloned into the
BamHI-HindIII site of vector pND-1 (a gift from
Dr. Didier Negre) to form expression plasmid pNDDI. This plasmid was
used to transform Escherichia coli BL21(DE3)pLysS by the
method of Chung et al. (16). Because attempts to express the
full-length dienoyl-CoA isomerase were unsuccessful, the cDNA of
the mature dienoyl-CoA isomerase was generated from plasmid pNDDI by
PCR using primer 5'-CAGGATCCCATATGAGCTCCTCTGCACAAGAGGCGT-3' for
introducing a starting methionine followed by three serines and a 3'
primer 5'-CAGTAAGCTTATCAGAGCTTGGAGAAGGTGATGCTT-3'. BamHI and
HindIII restriction sites were introduced with the 5' primer and 3' primer, respectively. The PCR product was subcloned into pGEM-T
Easy vector and transformed into E. coli JM109 as described by the manufacturer. The plasmid pGEM with the insert was isolated from
transformants and digested with BamHI and
HindIII. The DNA fragments corresponding to the dienoyl-CoA
isomerase was obtained by GeneClean and ligated into expression vector
pND-1. The expression construct, designated as pNDdi, was used to
transform E. coli strain BL21(DE3)pLysS. The transformants
were grown in LB medium to an absorbance of about 1.0 at 600 nm and
then induced by 0.6 mM
isopropyl-1-thio- Site-directed Mutagenesis of Dienoyl-CoA
Isomerase--
Site-directed mutagenesis was carried out by use of a
Transformer site-directed mutagenesis kit
(CLONTECH) following the manufacturer's instruction. A synthetic oligonucleotide,
5'-ATGCTTCAATAAGATTGAAAAAGGAAG-3', designed to eliminate a
SspI site, was used as the selection primer. The following
synthetic oligonucleotides were used as the mutagenic primers. The
substituting nucleotide is underlined, and the mutant codon
is in boldface: Asp176
The selection primer and one of the mutagenic primers were
simultaneously annealed to the template of the denatured
double-stranded pNDdi and then incorporated into a new strand of DNA as
a result of the elongation catalyzed by T4 DNA polymerase. After
digestion with SspI, the mixture of parent and newly
synthesized DNA was transformed into E. coli BMH 71-18 mutS. The plasmids were isolated from the transformants and
digested again with SspI. The digestion mixture was
transformed into E. coli BL21(DE3)pLysS, and the desired mutant was selected for the absence of the SspI restriction
site. The point mutation was confirmed by sequencing of the respective mutant strain. Expression of the mutant enzymes was achieved by the
procedure used for expressing the wild-type dienoyl-CoA isomerase.
Purification of Recombinant Wild-type and Mutant Dienoyl-CoA
Isomerase--
The frozen pellet from ~350 ml of cell culture was
suspended in 10 ml of 10 mM potassium Pi (pH
8.8) containing 5 mM mercaptoethanol, 1 mM
EDTA, 1 mM benzamidine, and 0.5 mM PMSF (buffer
A) and sonicated 12 times for 20 s each. The resultant suspension
was centrifuged at 100,000 × g for 30 min. The
supernatant was loaded onto a Q-Sepharose column (1.5 × 17 cm)
previously equilibrated with buffer A. The column was extensively
washed with buffer A and then eluted with a gradient made up of 120 ml
of buffer A and 120 ml of buffer A containing 0.4 M KCl.
The active fractions were combined and concentrated in an Amicon
concentrator with a PX-10 membrane. The concentrate was diluted 10-fold
with 10 mM potassium Pi (pH 6.0) containing 1 mM EDTA, 5 mM mercaptoethanol, and 20%
glycerol (buffer B) and applied to an S-Sepharose column (1.5 × 4 cm) previously equilibrated with buffer B. After washing extensively
with buffer B, the column was developed with a gradient made up of 30 ml of buffer B and 30 ml of buffer B containing 0.4 M KCl.
The active fractions were combined and concentrated.
CD Spectra of Wild-type and Mutant Dienoyl-CoA
Isomerases--
Far-UV CD scans were acquired between 190 and 250 nm
with an AVIV CD spectrophotometer equipped with temperature control. Two average scans were acquired at 20 °C for each sample. The scans
were normalized for protein concentration and corrected for the
influence of the buffer.
Molecular Characterization of the Mitochondrial and
Peroxisomal Forms of Dienoyl-CoA Isomerase--
For a planned
mechanistic study of rat dienoyl-CoA isomerase, milligram quantities of
highly active enzyme were required. Although a recombinant form of this
enzyme, lacking its 53 N-terminal amino acid residues, has been
described (6), its activity was much lower than that of the native
enzyme and too low for the contemplated mechanistic study. Attempts to
express the full-length isomerase-cDNA were unsuccessful. Hence, we
embarked on the molecular characterization of the native mitochondrial
and peroxisomal dienoyl-CoA isomerases with the aim of producing a
recombinant form of the highly active mature enzyme.
N-terminal sequencing of the purified rat liver dienoyl-CoA isomerase
revealed the presence of several polypeptides in agreement with the
observation of at least three closely spaced bands when the same
preparation was subjected to SDS-PAGE (Fig.
1A, lanes 2 and
5). Because this preparation may have been a mixture of the
mitochondrial and peroxisomal forms of dienoyl-CoA isomerase, the
enzyme was also purified from rat hearts, which contain few peroxisomes. SDS-PAGE and immunoblotting of the heart preparation led
to the identification of the dienoyl-CoA isomerase, which seemed to be
slightly larger than the liver forms of the enzyme (Fig. 1,
A and B, lanes 2 and 3).
When the heart dienoyl-CoA isomerase was subjected to N-terminal
sequencing, a unique sequence was obtained for the first 20 residues
(Fig. 2A). The sequence,
beginning with 3 serine residues, perfectly matched the predicted amino acid sequence of rat liver dienoyl-CoA isomerase (6) from residue 35 through residue 54. The missing residues 1 through 34 constitute a
polypeptide that has the properties of a mitochondrial targeting sequence (Fig. 2B). This conclusion is supported by the
following properties of this polypeptide: It is rich in positively
charged and hydroxylated residues (3 arginine, 1 lysine, 4 serine, and 3 threonine residues), and it is devoid of acidic residues and does not
have a large stretch of uncharged residues (Fig. 2B). Moreover, its N terminus forms a positively charged amphiphilic helix
(Fig. 2B). The mitochondrial precursor protein seems to have
only one cleavage site. Another potential cleavage site that is
indicated by a hydrophilic residue and a serine at positions 27 and 30, respectively, is not susceptible to proteolysis, due to the absence of
an arginine residue from position 25.
In an effort to characterize the peroxisomal dienoyl-CoA isomerase, the
enzyme was isolated from purified rat liver peroxisomes. A better than
80-fold purification was achieved by chromatography on hydroxylapatite
followed by chromatofocusing. The resultant preparation was composed of
at least three proteins (Fig. 1A, lane 4). The
major component with a molecular mass of 32 kDa was identified by
immunoblotting as dienoyl-CoA isomerase (Fig. 1B, lane
4). N-terminal sequencing of the material corresponding to the
32-kDa band revealed the presence of several forms of dienoyl-CoA isomerase with different N termini. The N termini of the two most prominent polypeptides were located to positions 37 and 39 of the
full-length protein (Fig. 2A). In an attempt to determine whether the peroxisomal dienoyl-CoA isomerase exists in vivo
as a truncated protein, purified peroxisomes were incubated with boiling SDS incubation buffer and subjected to SDS-PAGE followed by
immunoblotting with antibodies purified by affinity chromatography on a
dienoyl-CoA isomerase-Sepharose column. As shown in Fig. 1C,
only one band corresponding to a 32-kDa protein was observed. Thus, it
seems that the native peroxisomal dienoyl-CoA isomerase is a truncated
protein with a ragged N terminus.
Mechanistic Study of Dienoyl-CoA Isomerase by Site-specific
Mutagenesis--
The cDNA coding for dienoyl-CoA isomerase was
cloned from a rat liver cDNA library by PCR. However, attempts to
express the full-length protein in E. coli were
unsuccessful. We therefore generated the cDNA for the mature rat
heart isomerase from the full-length cDNA by PCR. This mature
mitochondrial form of dienoyl-CoA isomerase was successfully expressed
in E. coli. A 10-fold purification of dienoyl-CoA isomerase,
beginning with a soluble extract of such cells, yielded the pure enzyme
in 65% yield. This enzyme preparation exhibited an activity of 960 units/mg (Table I), which is
significantly higher than the activity of the enzyme isolated from rat
liver (3). The fact that the recombinant dienoyl-CoA isomerase also
exhibited trienoyl-CoA isomerase activity, proved that both catalytic
properties are associated with the same protein.
For the planned mechanistic study, mutant proteins were needed with
Asp204, Glu196, and Asp176 replaced
by uncharged amino acid residues. The mutations shown in Table I were
introduced by site-specific mutagenesis, and the recombinant mutant
proteins were purified to apparent or near homogeneity as indicated by
SDS-PAGE (results not shown). The near-UV CD spectra of all mutant
proteins were virtually indistinguishable from the spectrum of the
wild-type enzyme (data not shown). When assayed for 3,5
In an effort to further explore the mechanism of
dienoyl-CoA/trienoyl-CoA isomerase, its activity with
2-trans,5-cis-octadienoyl-CoA as a substrate was
evaluated. With the wild-type enzyme, a small but significant
conversion of the 2,5 diene to the 2,4 isomer was observed (Table I).
The rate of the 2,5
The Asp204 mutants also catalyzed a slow but detectable
2,5
The question of whether the differences between the reaction rates
observed with various mutants and substrates were due to changes in
Km values, Vmax values, or
both were addressed. The kinetic parameters listed in Table
II clearly show that the Km values varied little and that differences between Vmax values were the major cause of rate
differences observed at fixed substrate concentrations of 20 µM.
The main reason for the molecular characterization of the mature
dienoyl-CoA isomerases was the need to produce a highly active recombinant form of this enzyme. Such enzyme had not been obtained when
recombinant versions of the full-length protein and of an artificially
truncated isomerase were generated (6). However, this study was also
prompted by a desire to demonstrate unambiguously the dual location of
this enzyme in mitochondria and peroxisomes. The presence of the same
dienoyl-CoA isomerase in both organelles had been suspected when
antibodies raised against the mitochondrial enzyme were found to
cross-react with the peroxisomal isomerase (5). Cloning of dienoyl-CoA
isomerase had revealed the presence of a peroxisomal targeting signal
and an N terminus with properties of mitochondrial targeting sequence.
Moreover, antibodies raised against a synthetic peptide corresponding
to the C terminus of the enzyme recognized 32-kDa and 36-kDa proteins
in mitochondria and peroxisomes, respectively (6).
This study, besides revealing the N termini of the mature forms
of dienoyl-CoA isomerase, confirms the dual localization of the enzyme
to mitochondria and peroxisomes. However, in contrast to a previous
report (6), the peroxisomal dienoyl-CoA isomerase was found to have a
molecular mass of 32 kDa and hence to be a truncated form of the
full-length protein. The removal of N-terminal sequences similar in
size to the mitochondrial targeting sequence could be a consequence of
the susceptibility of this region of the protein to proteolysis. This
idea has not been tested nor have other explanations been ruled out.
However, the detection of only one band corresponding to a 32-kDa
protein when intact peroxisomes were solubilized with boiling SDS
incubation buffer and then subjected to SDS-PAGE and immunoblotting,
supports the conclusion that the truncated form(s) of the peroxisomal
dienoyl-CoA isomerase is(are) present in vivo and are not
artifacts of the isolation procedure. This study additionally
demonstrates that the heart and liver enzymes are identical over a
stretch of 20 amino acid residues. Hence, both enzymes are most likely
products of the same gene.
The successful expression of the mature cardiac dienoyl-CoA isomerase
in E. coli achieved two goals. Foremost, a highly active form of this enzyme became available. In fact, the maximal specific activity of 2450 units/mg for the recombinant isomerase was 6 times
higher than the Vmax of the enzyme isolated from
rat liver (3). Although this difference may be due in part to the use of HPLC-purified substrate in this study, it also reflects the preparation of a purer enzyme. In any case, the recombinant enzyme exhibited an activity well suited for the planned mechanistic study.
The second achievement was the demonstration that both dienoyl-CoA
isomerase and trienoyl-CoA isomerase are associated with the same
protein. This result puts to rest any existing suspicion that the two
activities may be expressions of distinct but similar proteins.
The successful creation and purification of several mutant forms of
dienoyl-CoA isomerase permitted an analysis of its catalytic mechanism.
Dramatic activity decreases were observed as the result of replacing
Asp204 and Glu196 with neutral amino acids.
This finding supports the proposed function of these residues in proton
transfers from and to the substrate (7), because it agrees with the
general prediction that the mutation of a residue that directly
participates in a reaction as a general acid/base would be expected to
cause a 105 or greater decrease in activity (17).
The use of 2,5-octadienoyl-CoA as a substrate analog revealed slow, but
measurable 2,53,5,
2,4-Dienoyl-CoA
isomerase (DI), an auxiliary enzyme of unsaturated fatty acid
-oxidation, was purified from rat mitochondria and peroxisomes and
subjected to N-terminal sequencing to facilitate a mechanistic study of
this enzyme. The mature mitochondrial DI from rat heart was lacking its
34 N-terminal amino acid residues that have the properties of a
mitochondrial targeting sequence. The peroxisomal isomerase was
identified as a product of the same gene with a truncated and ragged N
terminus. Expression of the cDNA coding for the mature
mitochondrial DI in Escherichia coli yielded an enzyme
preparation that was as active as the native DI. Because the
recombinant DI also exhibited
3,5,7,
2,4,6-trienoyl-CoA isomerase (TI)
activity, both isomerases reside on the same protein. Mutations of any
of the 3 acidic amino acid residues located at the active site (Modis,
Y., Filppula, S. A., Novikov, D. K., Norledge, B., Hiltunen,
J. K., and Wierenga, R. K. (1998) Structure 6, 957-970) caused activity losses. In contrast to only a 10-fold
decrease in activity upon replacement of Asp176 by
Ala, substitutions of Asp204 by Asn and of
Glu196 by Gln resulted in 105-fold lower
activities. Such activity losses are consistent with the direct
involvement of these latter two residues in the proposed proton
transfers at carbons 2 and 6 or 8 of the substrates. Probing of the
wild-type and mutants forms of the enzyme with 2,5-octadienoyl-CoA as
substrate revealed low
2,
3-enoyl-CoA isomerase and
5,
4-enoyl-CoA isomerase activities
catalyzed by Glu196 and Asp204, respectively.
Altogether, these data reveal that positional isomerizations of the
diene and triene are facilitated by simultaneous proton transfers
involving Glu196 and Asp204, whereas each
residue alone can catalyze, albeit less efficiently, a monoene isomerization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation
requires several auxiliary enzymes in addition to the enzymes necessary for the breakdown of saturated fatty acids (for a review see Ref. 1).
One of the auxiliary enzymes is
3,5,
2,4-dienoyl-CoA isomerase
(dienoyl-CoA isomerase),1
which catalyzes the isomerization of 3,5-dienoyl-CoA to 2,4-dienoyl-CoA (2, 3). During the
-oxidation of unsaturated fatty acids with
odd-numbered double bonds, 5-enoyl-CoA intermediates can be converted
to 3,5-dienoyl-CoA by the sequential actions of acyl-CoA dehydrogenase
and
3,
2-enoyl-CoA isomerase (EC 5.3.3.8)
(enoyl-CoA isomerase). The further degradation of 3,5-dienoyl-CoA
requires its isomerization to 2,4-dienoyl-CoA, because the latter
compound can be reduced by 2,4-dienoyl-CoA reductase (EC 1.3.1.34) to
3-enoyl-CoA. The isomerization of 3-enoyl-CoA to 2-enoyl-CoA by
enoyl-CoA isomerase completes the reductase-dependent
sequence of reactions during the
-oxidation of unsaturated fatty
acids with odd-numbered double bonds.
3,5,7,
2,4,6-trienoyl-CoA isomerase
(trienoyl-CoA isomerase), an enzyme involved in the degradation of
unsaturated fatty acids with conjugated double bonds, was found to be a
component enzyme of dienoyl-CoA isomerase (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3,5,
2,4-dienoyl-CoA isomerase,
3,5-cis-octadienoyl-CoA, 300 nm, 27,800 M
1 cm
1;
3,5,7,
2,4,6-trienoyl-CoA isomerase,
3,5,7-decatrienoyl-CoA, 337 nm, 49,300 M
1
cm
1;
2,5,
2,4-enoyl-CoA
isomerase, 2,5-cis-octadienoyl-CoA, 300 nm, 27,800 M
1 cm
1;
2,5,
3,5-enoyl-CoA isomerase,
2,5-cis-octadienoyl-CoA, 237 nm, 16,250 M
1 cm
1;
3,
2-enoyl-CoA isomerase,
3-cis-octenoyl-CoA, 263 nm, 6,700 M
1 cm
1. One unit of enzyme
activity is defined as the amount of enzyme that catalyzes the
conversion of 1 µmol of substrate to product in 1 min. Kinetic
parameters (Km and Vmax) were
obtained by nonlinear curve fitting using SigmaPlot 2000. Protein
concentrations were determined as described by Bradford (10) with
bovine serum albumin as standard.
-D-galactopyranoside for 4 h.
Cells were harvested by centrifugation at 3000 × g for
5 min and stored at
80 °C.
Ala,
5'-GGAGGCGTGGCTCTTATTTCTG-3'; Asp176
Asn,
5'-GGAGGCGTGAATCTTATTTCTG-3';
Glu196
Ala,
5'-CCAAGTCAAGGCGGTGGATGTG-3; Glu196
Asp,
5'-CCAAGTCAAGGATGTGGATGTGG-3'; Glu196
Gln,
5'-CCAAGTCAAGCAGGTGGATGTG-3';
Asp204
Ala,
5'-CTGGCTGCTGCTGTAGGAACGCTG-3'; Asp204
Asn,
5'-CTGGCTGCTAATGTAGGAACGC-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
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Fig. 1.
SDS-PAGE and immunoblotting of rat
dienoyl-CoA isomerases (DI). A, after
SDS-PAGE and staining with Coomassie Brilliant Blue: Molecular mass
standards (lane 1), rat DI purified from liver (lanes
2 and 5), partially purified DI from rat heart
(lane 3), and partially purified DI from rat liver
peroxisomes (lane 4). B, immunoblot of the above
DI's with antiserum to DI. C, immunoblot of partially
purified DI from rat liver peroxisomes (lane 6) and of
purified rat liver peroxisomes (lanes 7 and 8)
using purified monospecific antibodies to DI.
View larger version (20K):
[in a new window]
Fig. 2.
N-terminal sequences of mature rat
dienoyl-CoA isomerases and sequence of the mitochondrial targeting
peptide. A, N-terminal sequences of rat heart
mitochondrial dienoyl-CoA isomerase and of two forms of rat liver
peroxisomal dienoyl-CoA isomerases. The arrows mark the N
termini of the sequences, and the numbers indicate the
position of residues in the full-length protein. B, the
mitochondrial targeting sequence of rat heart dienoyl-CoA isomerase.
The positions of basic and hydroxylated amino acid residues are
indicated by and *, respectively. The amphiphilic helix formed by
the first 12 residues of the leader peptide is shown as a helical
wheel. Hydrophobic residues are framed.
Activities of wild-type and mutant dienoyl-CoA isomerases
2,4
dienoyl-CoA isomerase activity, the replacement of either
Asp204 or Glu196 by a neutral amino acid
residue resulted in an ~105-fold lower activity (Table
I). This observation agrees with the proposed functions of these two
residues in the direct proton transfer to or from the substrate (7).
Substitution of Glu196 by an aspartate residue produced a
mutant enzyme that retained ~3% of the isomerase activity (Table I).
This lower but significant activity demonstrates that the
-carboxyl
group of Asp196 can facilitate the proton transfer although less
efficiently than the
-carboxyl group of Glu196. The
mutation of the third acidic group at the active site,
Asp176, to Ala caused a 10-fold decrease in activity. The
limited effect of this mutation argues against a direct participation
of this residue in catalysis. The effects of mutating
Asp204, Glu196, and Asp176 on the
trienoyl-CoA isomerase activity of this enzyme were comparable to the
impact on the dienoyl-CoA isomerase except that the activity losses due
to the D176A and E196D mutations were more severe (Table I). Overall
these data indicates that the active site of dienoyl-CoA isomerase is
identical with the active site of trienoyl-CoA isomerase and that the
same acidic residues, Glu196 and Asp204,
catalyze the proton transfers that result in the 3,5
2,4 and 3,5,7
2,4,6 isomerizations.
2,4 conversion was more than 104
times slower than the 3,5
2,4 isomerization. Surprisingly, mutants of Glu196 were more active than the wild-type enzyme in
catalyzing this reaction (Table I). The E196Q mutant, which was 10 times as active as the wild-type isomerase, permitted a spectroscopic
analysis of the 2,5
2,4 isomerization. The time-dependent
spectral changes shown in Fig.
3B are indicative of a direct
2,5
2,4 isomerization rather than a sequential 2,5
3,5
2,4
conversion. These spectra do not provide evidence for the formation of
a 3,5 intermediate with an absorbance maximum at 238 nm nor do they
reveal a 3,5
2,4 isomerization as shown in Fig. 3A.
Because the 2,5
2,4 conversion catalyzed by the E196Q mutant was 60 times faster than the 3,5
2,4 isomerization catalyzed by the same
enzyme (Table I), the 2,5
2,4 isomerization seems to be a one-step
conversion.
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Fig. 3.
Spectrophotometric analyses of the
isomerizations of 3,5-octadienoyl-CoA and 2,5-octadienoyl-CoA catalyzed
by dienoyl-CoA isomerase. A, spectral changes
associated with the isomerization of 3.5-octadienoyl-CoA to
2,4-octadienoyl-CoA (3,5 2,4) catalyzed by
wild-type dienoyl-CoA isomerase. Spectrum 1 at time 0; spectra 2-4
were recorded 20 s, 1.5 min, and 10 min after the addition of
enzyme. B, spectral changes associated with the
isomerization of 2,5-octadienoyl-CoA to 2,4-octadienoyl-CoA
(2,5
2,4) catalyzed by the E196Q mutant of
dienoyl-CoA isomerase. Spectrum 1 at time 0; spectra 2-4 were recorded
20 s, 1.5 min, and 20 min after the addition of enzyme.
2,4 isomerization. However, as shown in Fig.
4C, the formation of the 2,4 isomer proceeded with a lag. Spectral analyses of the reactions that
occurred during (Fig. 4C, period A) and after the lag phase (Fig. 4C, period B) revealed an initial
2,5
3,5 isomerization indicated by an increase in the absorbance at
238 nm due to the formation of the 3,5 diene (Fig. 4A)
followed by the formation of the 2,4 isomer detected at 300 nm (Fig.
4B). Overall, the product formation occurred by a
2,5
3,5
2,4 conversion that showed a pronounced lag in the
formation of the 2,4 isomer, because the first reaction proceeded
faster than the second reaction (Table I). Because the D204A mutant
catalyzed the 2,5
3,5 conversion, it was expected to catalyze also
the isomerization of 3-octenoyl-CoA to 2-octenoyl-CoA. This conversion
was in fact observed and found to take place at a rate of 0.034 unit/mg
as compared with 0.4 unit/mg for the 2,5
3,5 isomerization.
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Fig. 4.
Spectrophotometric analysis of the
isomerization of 2,5-octadienoyl-CoA to 2,4-octadienoyl-CoA catalyzed
by the D204A mutant of dienoyl-CoA isomerase. Spectral changes
observed during (A) the initial phase of the isomerization
reaction (2,5 3,5), (B) the later
phase of the isomerization reaction (3,5
2,4),
and (C) absorbance change at 300 nm observed during the
isomerization of 2,5-octadienoyl-CoA to 2,4-octadienoyl-CoA catalyzed
by the D204A mutant as a function of time.
Kinetic parameters of wild-type and mutant dienoyl-CoA isomerases
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,4 isomerizations. Because the different positional
isomers of octadienoyl-CoA have distinct UV spectra, it was possible to
analyze the mechanisms of these isomerizations. The spectral changes
observed with mutant E196Q were suggestive of a direct 2,5
2,4
isomerization without the formation of an intermediate. The only
alternative route, via a sequence of isomerizations with
3,5-octadienoyl-CoA as an intermediate, was ruled out because the
3,5
2,4 isomerization was much slower than the overall 2,5
2,4 isomerization. Hence the observed 2,5
2,4 isomerization must be the
result of a 5
4 double-bond shift as shown in Fig.
5. Asp204 is the obvious
candidate to facilitate this monoene isomerization by catalyzing
a 1,3-proton shift from carbon 4 to carbon 6. Such mechanism for single
double-bond isomerizations has been proposed for cholesterol
oxidase (18) and
3,
2-enoyl-CoA isomerase
(19) based on observed intramolecular 1,3-hydrogen shifts. If
mechanistically similar, the 1,3-proton shift catalyzed by
Asp204 may not be concerted, as shown in Fig. 5, but rather
proceed in two steps by removal of a proton from carbon 4 and formation of a stabilized carbanion followed by addition of a proton to carbon 6. Noteworthy is the observation that the 2,5
2,4 isomerization catalyzed by mutant E196Q proceeded 11 times faster than the same reaction catalyzed by the wild-type isomerase. The lower rate detected
with the wild-type enzyme may be due to a higher pK value of
Asp204 induced by Glu196. Such electrostatic
effect on Asp204 would not be effective in the E196Q
mutant.
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Fig. 5.
Proposed catalytic mechanisms of mutant E196Q
for the isomerizations of 2,5-dienoyl-CoA (2,5) to
2,4-dienoyl-CoA (2,4) and of mutant D204A for the
isomerizations of 2,5-dienoyl-CoA (2,5) to
3,5-dienoyl-CoA (3,5).
The 2,52,4 isomerization catalyzed by mutant D204A was more
complex than the conversion brought about by the E196Q mutant. The
progress curve for the D204A-catalyzed 2,5
2,4 isomerization showed a
lag that was shown to correspond to the conversion of 2,5-octadienoyl-CoA to its 3,5 isomer. Because the 2,5
3,5
isomerization was faster than the subsequent 3,5
2,4 isomerization,
the 3,5 intermediate accumulated and initially was detectable. The
formation of 3,5-octadienoyl-CoA was the result of a double-bond shift
from carbon 2 to carbon 3. This double-bond isomerization must have been catalyzed by Glu196, which is proposed to facilitate a
1,3-proton shift from carbon 4 to carbon 2 (Fig. 5). Again, the proton
transfers may not be concerted as shown in Fig. 5 but may be
sequential, resulting in the formation of a carbanion intermediate. An
alternative route with a carbocationic intermediate represents an
unlikely mechanism.
The analyses of the 2,52,4 isomerizations provide good
evidence for Glu196 being close to carbon 2 and
Asp204 close to carbon 6 as shown in Fig.
6. Both residues are necessary for the
3,5
2,4 isomerization that proceeds by a simultaneous shift of both
double bonds (3). A similar mechanism is envisioned for the triene
isomerization except that Asp204 must be close to carbon 8 (Fig. 6). Such a dual role of Asp204 suggests a certain
flexibility of the residue and/or requires different positioning of the
dienoyl-CoA and trienoyl-CoA substrates at the active site. Either way,
the function of Asp204 in the isomerization of the triene
comes at a price that is reflected by the almost 50-fold lower activity
of trienoyl-CoA isomerase as compared with dienoyl-CoA isomerase.
|
Altogether, this study demonstrates the need for two acidic residues to
facilitate the proton transfers that result in positional isomerizations of dienes and trienes. In contrast, proton transfers that cause monoenes to shift by one carbon only require a single acidic
residue as previously documented for other isomerases (18, 19).
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FOOTNOTES |
---|
* This work was supported by U.S. Public Health Service Grant HL30847 from the NHLBI, National Institutes of Health and by Grant RR03060 to Research Centers of Minority Institutions.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Chemistry, City College of the City University of New York, Convent
Ave. at 138th St., New York, NY 10031. Tel.: 212-650-8323; Fax:
212-650-8322; E-mail: hoschu@sci.ccny.cuny.edu.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011315200
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ABBREVIATIONS |
---|
The abbreviations used are:
dienoyl-CoA
isomerase or 3, 52,4-dienoyl-CoA isomerase,
3,5,
2,4-dienoyl-CoA isomerase;
enoyl-CoA
isomerase,
3,
2-enoyl-CoA isomerase;
CD, circular dichroism;
HPLC, high performance liquid chromatography;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
PMSF, phenylmethylsulfonyl fluoride;
trienoyl-CoA isomerase,
3,5,7,
2,4,6-trienoyl-CoA isomerase.
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REFERENCES |
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