Very long-chain acyl-CoA dehydrogenase (VLCAD) is
one of four flavoproteins which catalyze the initial step of the
mitochondrial
-oxidation spiral. By sequence comparison with other
acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the
catalytic residue that abstracts the
-proton in the

-dehydrogenation reaction. Replacing Glu-422 with glutamine
(E422Q) caused a loss of enzyme activity by preventing the formation of
a charge transfer complex between VLCAD and palmitoyl-CoA. This result
provides further evidence for Glu-422 being part of the active site of VLCAD.
F418L is a disease-causing mutation in human VLCAD deficiency. Unlike
wild-type VLCAD, F418L and F418V contained no bound FAD when expressed
at extremely high levels in the baculovirus expression system. Although
F418T and F418Y bound FAD at a level similar to that of wild-type
VLCAD, both showed reduced Vmax values toward
palmitoyl-CoA, most likely due to a decrease in the rate of
enzyme-bound FAD reduction. These data suggest that Phe-418 is involved
in the binding and subsequent reduction of FAD. FAD-deficient VLCADs
(F418L, F418V, and apo-VLCAD) showed increased sensitivity to
trypsinization. Loss of FAD may change the folding of VLCAD subunit.
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INTRODUCTION |
Mitochondrial fatty acid
-oxidation is one of the main
energy-yielding metabolic pathways in eukaryotes. The initial step in
the mitochondrial fatty acid
-oxidation spiral is catalyzed by four
acyl-CoA dehydrogenases which have different, but overlapping, substrate chain length specificities. Very long-chain acyl-CoA dehydrogenase (VLCAD; EC
1.3.99.13),1 a novel
mitochondrial inner membrane-associated acyl-CoA dehydrogenase, shows
activity toward CoA esters of long- and very long-chain fatty acids
(1). In human tissues, VLCAD accounts for the majority (
80%) of
palmitoyl-CoA dehydrogenation activity (2). VLCAD is thought to play an
important role in the initial
-oxidation cycles of long-chain fatty
acids, along with enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein, which catalyzes the succeeding three reactions (3, 4).
Short- (SCAD), medium- (MCAD), and long- (LCAD) chain acyl-CoA
dehydrogenases are homotetramers of approximately 40-kDa polypeptides which contain 4 mol of FAD/mol of enzyme (5, 6), whereas VLCAD is a
homodimer of a 71-kDa polypeptide which contains 2 mol of FAD/mol of
enzyme (1). SCAD, MCAD, and LCAD share a high degree of sequence
similarity throughout their entire sequences (7). Although VLCAD is
highly homologous to other acyl-CoA dehydrogenases at its amino
terminus, its carboxyl terminus contains a long tail of approximately
180 amino acid residues not shared by other acyl-CoA dehydrogenases (8,
9).
In the 
-dehydrogenation reaction, the abstraction of the
-proton from the acyl-CoA substrate is catalyzed by an acidic residue in acyl-CoA dehydrogenase and is followed by the transfer of
the
-hydride to the N-5 position of the enzyme-bound FAD (10, 11).
In MCAD, Glu-376 has been determined to be the catalytic residue that
abstracts the
-proton by mutational (12) and x-ray crystallographic
analyses (13). The region surrounding Glu-376 of MCAD shows significant
sequence similarity to other acyl-CoA dehydrogenases (Fig.
1). This glutamate residue is conserved
in both VLCAD and SCAD. Recently, Glu-368 of SCAD was shown to be a
catalytic residue by mutational analysis (14). On the other hand, in
LCAD, glutamate is not conserved at the position corresponding to
Glu-376 of MCAD, and instead Glu-261 has been identified as a catalytic
residue (15). The presence of a glycine residue in VLCAD at the
position corresponding to Glu-261 of LCAD makes it more likely that
Glu-422 of VLCAD is a catalytic residue.

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Fig. 1.
Sequence alignment of acyl-CoA
dehydrogenases. The arrow indicates the position of the
phenylalanine residue (F) of VLCAD which is replaced by a
leucine (L) in a patient with VLCAD deficiency. The
dot indicates the position of the catalytic residue, Glu-376, of MCAD.
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VLCAD deficiency is a newly reported disorder of fatty acid metabolism
that frequently leads to hypertrophic cardiomyopathy and sudden death
in infancy (16-20). To date, 13 patients with this disease have been
diagnosed and characterized at the molecular level (9, 20-22).
Mutations identified in the VLCAD gene have been heterogenous with most
causing rapid degradation of the mRNA, protein, or both (21).
Recently, we found a disease-causing mutation, F418L (precursor
position 458), in a patient with VLCAD deficiency in whom a
cross-reactive protein of normal size was detected by immunoblot analysis with anti-VLCAD
antibody.2 We have used the
baculovirus expression system and site-directed mutagenesis to
investigate the roles of Phe-418 and Glu-422 in the enzymatic activity
of VLCAD. Our results suggest specific catalytic functions for Phe-418
and Glu-422 in the active site of VLCAD.
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EXPERIMENTAL PROCEDURES |
Materials--
VLCAD was purified from human liver as described
(2). Human VLCAD cDNA was cloned previously (9). A
TransformerTM Site-Directed Mutagenesis Kit was purchased
from CLONTECH (Palo Alto, CA). MaxBac®
Baculovirus Express Vector System Kit was purchased from Invitrogen
(San Diego, CA). Acyl-CoAs were synthesized by the mixed anhydride
method (23) and purified by DEAE-cellulose column chromatography (24).
Phenazine ethosulfate (PES) and bovine spleen trypsin were purchased
from Boehringer Mannheim (Germany).
Site-directed Mutagenesis and Vector Construction--
VLCAD
cDNA was polymerase chain reaction-amplified and subcloned into
pT7Blue T-Vector (pT7B-VLCAD). HindIII and BamHI
fragments from pT7B-VLCAD were independently subcloned into
corresponding restriction sites of the baculovirus transfer vector,
pBlueBacIII (pBBIII-VLCAD).
Site-directed mutagenesis was performed by the method of Deng and
Nickoloff (25) using a TransformerTM Site-Directed
Mutagenesis Kit. Mutant cDNAs were synthesized according to the
manufacturer's instructions, using pT7B-VLCAD as template.
BamHI fragments of the mutant pT7B-VLCAD cDNAs were inserted into the BamHI site of pBBIII-VLCAD.
Preparation of Recombinant Baculoviruses--
Recombinant
baculovirus transfer vectors were co-transfected into Sf9 cells with
wild-type Autographa californica nuclear polyhedrosis virus
DNA by cationic liposome-mediated transfection. Cell transfection,
plaque assays, isolation of plaque purified viruses, and preparation of
high-titer stocks were performed as described in the manufacturer's
instructions.
Expression and Purification of Variant VLCADs--
Sf9
cells (approximately 5 × 108 cells) were infected
with recombinant viruses as multiplicity of infection
3. On day 3 after infection, the cells were collected, washed twice with
phosphate-buffered saline, and resuspended in 30 ml of 0.25 M sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride.
The suspension was homogenized using a Potter homogenizer and
centrifuged at 1,500 × g for 5 min. The supernatant
was further centrifuged at 10,000 × g for 10 min, and
the mitochondrial pellet was obtained. Expressed protein was extracted
from the mitochondrial fraction and purified by phosphocellulose and
DEAE-cellulose column chromatography as described previously (1).
Approximately 0.5 mg of the expressed proteins were obtained and stored
in 0.5 ml of 50% glycerol, 50 mM potassium phosphate (pH
7.5) at
20 °C.
FAD Content--
Fifty µl of the partially purified sample
(approximately 50 µg) was diluted with 350 µl of 20 mM
potassium phosphate (pH 7.5), and then 25 µl of 50% trichloroacetate
was added. The sample was incubated on ice for 30 min and then
centrifuged at 10,000 × g for 5 min. Three
hundred-fifty µl of the supernatant was transferred to a new tube,
diluted with 100 µl of water, and 50 µl of NaOH (1 N)
was added to adjust the pH to 2.8. The fluorescence intensity of the
solution (excitation at 445 nm and emission at 520 nm) was measured
using a fluorometer (Hitachi F-2000). The FAD content was calculated
using standard FAD (Sigma).
Enzyme Assay--
VLCAD enzyme activity was measured by the
dye-reduction method using PES as an electron transfer dye and
dichloroindophenol as an electron acceptor. The reaction mixture
contained 50 mM potassium phosphate (pH 7.5), 30 µM acyl-CoA, 35 µM dichloroindophenol, 1 mM N-ethylmaleimide, 3 mM PES, and
enzyme. The reaction was initiated by adding 3 mM PES, and
the reduction of dichloroindophenol was monitored by the decrease in
absorbance at 600 nm. The activity was calculated using a molar
extinction coefficient of 21,000 M
1
cm
1.
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RESULTS |
Expression of Wild-type VLCAD by the Baculovirus Expression
System--
The cDNA-directed expression of wild-type VLCAD was
performed using the baculovirus expression system. A recombinant
baculovirus containing full-length VLCAD cDNA with leader peptide
sequence was used to infect Sf9 cells. Immunoblot analysis of
the cells infected with recombinant baculovirus revealed a large amount of the precursor protein along with the mature form (Fig.
2A). Subcellular fractionation
showed that the expressed mature VLCAD was associated with mitochondria
while the precursor protein was detected in both the mitochondrial and
microsomal fractions (Fig. 2B). The precursor protein was
separated from the mature form by phosphocellulose column
chromatography.

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Fig. 2.
Expression of VLCAD by the baculovirus
expression system. A, immunoblot analysis of VLCAD in
Sf9 cells infected with VLCAD recombinant virus. 1 µg of cell
lysate was analyzed per lane. Lane 1, non-infected
Sf9 cells; lane 2, cells infected with Autographa californica multiple nuclear polyhedrosis virus;
lane 3, cells infected with VLCAD recombinant virus. The
arrow indicates the position of human liver VLCAD.
B, subcellular fractionation of expressed VLCAD in
Sf9 cells. Cells infected with VLCAD recombinant virus were
homogenized in 0.25 M sucrose, 10 mM Tris-HCl
(pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride using a Potter homogenizer, and separated by centrifugation
into nuclear (N), mitochondrial (Mt), microsomal
(Ms), and cytosolic fractions (Cs). 0.5 mg of each fraction was analyzed by immunoblotting. The arrows
labeled P and M indicate the positions of the
precursor and mature forms, respectively. C, SDS-PAGE of
partially purified wild-type and mutant VLCADs. Lane 1,
wild-type VLCAD; lane 2, E422Q; lane 3, E422D;
lane 4, F418L; lane 5, F418T; lane 6,
F418Y; lane 7, F418V. The arrow indicates the
position of human liver VLCAD.
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The recombinant VLCAD migrated at the same position (70 kDa) as the
human liver enzyme by SDS-PAGE (Fig. 2C). The native
molecular mass of recombinant VLCAD was estimated to be 148 kDa by
size-exclusive column chromatography, indicating that the expressed
protein forms a dimer. The recombinant VLCAD showed absorption maxima
near 280, 370, and 450 nm characteristic of the spectrum of FAD. The
FAD content of the expressed protein was estimated to be approximately 2 mol/mol of dimer (Table I). The FAD
content and catalytic properties of the recombinant VLCAD (Table
II) were the same as those of the human
liver enzyme.
Glu-422 Mutants--
To determine whether Glu-422 is a catalytic
residue in VLCAD, two mutant proteins were prepared replacing Glu-422
with Gln or Asp (Fig. 2C). The native molecular masses,
absorption maxima, and FAD contents of expressed E422Q and E422D did
not differ significantly from those of wild-type (Table I). The enzyme
activity associated with E422Q was barely detectable with palmitoyl-CoA
(Table II) or other carbon chain length substrates (data not shown).
The Vmax value of E422D toward palmitoyl-CoA was
only 10% of that of wild-type, while the Km value
(Table II) and substrate chain length specificity (data not
shown) did not differ significantly from wild-type. The
Km value of E422D for the electron transfer dye,
PES, was also similar to that of wild-type (Table II).
Spectral data was used to provide evidence for the formation of a
charge transfer complex (26). UV visible spectra were monitored at
various concentrations of substrate added to the enzyme solution (Fig.
3). Titration of palmitoyl-CoA with VLCAD results in the quenching of absorbance at 450 nm and the appearance of
a new absorption band at 580 nm (Fig. 3A), which are
characteristic changes in the acyl-CoA dehydrogenase complex (27). A
15-20 nm blue shift of the 370-nm peak and a minor shift of the 450-nm peak were also observed. On the other hand, when palmitoyl-CoA was
titrated with E422Q, quenching of absorbance at 450 nm and significant
shifts of the absorption maxima were barely detectable (Fig.
3B). Only with 10 µM palmitoyl-CoA was slight
quenching of absorbance at 450 nm detected in E422D (Figs.
3C and 4).

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Fig. 3.
UV visible spectra of mutant VLCADs.
Partially purified proteins (approximately 2.5 µM) in 0.1 M potassium phosphate (pH 7.5), 0.2% Tween 20 were
degassed and left under nitrogen gas. The spectra in the absence
(bold lines) and the presence of 1, 3, 6, and 10 µM palmitoyl-CoA (dotted lines) were recorded 2 min after the addition of palmitoyl-CoA (panels A, E, and
F). Panels B and C show the spectra in
the absence and presence of 10 µM palmitoyl-CoA, and
panel D shows the spectra only in the absence of
palmitoyl-CoA.
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Fig. 4.
Palmitoyl-CoA-dependent quenching
of the absorption at 450 nm. , wild-type; , E422Q; ,
E422D; , F418T; , F418Y.
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Phe-418 Mutants--
The Phe-418 residue of VLCAD is not conserved
in other acyl-CoA dehydrogenases. The corresponding residues in SCAD,
MCAD, IVD, and LCAD are threonine, tyrosine, tyrosine, and glutamine, respectively (Fig. 1). To investigate the role of Phe-418 in VLCAD, four mutants of VLCAD were prepared, replacing this amino acid with
leucine, threonine, tyrosine, and valine.
The final preparations of two of the four Phe-418 mutants, F418L and
F418V, were colorless despite yields that were similar to that of
wild-type (Fig. 2C). On the other hand, the final
preparations of F418T and F418Y were yellow, the same color as
wild-type. The positions of the absorbance maxima of F418T and F418Y
were similar to those of wild-type, whereas F418L and F418V exhibited
only 1 absorbance maximum at 280 nm (Table I). The FAD contents of F418L and F418V were barely detectable even by fluorometric measurement after liberation from the proteins. The FAD contents of F418T and F418Y
were not significantly different from that of wild-type. Gel filtration
analysis confirmed that all of the Phe-418 mutants formed dimers.
The enzyme activities of F418L and F418V toward various carbon chain
length acyl-CoAs were hardly detectable (Table II). No increase in
enzyme activity was observed when FAD (final 20 µM) was
added to the F418L and F418V preparations. On the other hand, the
Vmax values of F418T and F418Y toward
palmitoyl-CoA were 68 and 25% of wild-type, respectively. The
Km values of F418Y for palmitoyl-CoA and PES were
similar to those of wild-type, while those of F418T were somewhat
higher than those of wild-type. The substrate chain length
specificities of F418T and F418Y were not significantly different from
that of wild-type (data not shown).
The UV visible spectra of F418T and F418Y were monitored at various
concentrations of palmitoyl-CoA. F418T strongly quenched absorbance at
450 nm even at 1 µM palmitoyl-CoA, reaching its maximum
at
3 µM (Figs. 3E and 4). In contrast, F418Y
showed less quenching of absorbance at 450 nm compared with wild-type
(Figs. 3F and 4). The estimated Kred
values (the substrate concentration causing half-maximum quenching of
the absorbance at 450 nm) of wild-type, F418T, and F418Y were 3.3, 2.1, and 4.9 µM, respectively. The estimated
max values (the maximum decrease in absorbance at 450 nm) were 0.027, 0.038, and 0.017, respectively.
Apo-VLCAD--
Apo-VLCAD was prepared by passing wild-type VLCAD
through a Sephadex G-25 column three times in the absence of FAD. The
final preparation contained 0.2 mol of FAD/mol of VLCAD dimer. The
palmitoyl-CoA dehydrogenase activity was 0.75 units/mg, which was
increased to 4.5 units/mg by addition of 20 µM FAD to the
preparation. These results suggested that approximately 10% of VLCAD
in the preparation was of the holo-type. The UV visible spectra of
apo-VLCAD was similar to that of F418L (data not shown).
Wild-type, mutant, and apo-VLCADs were treated with various amounts of
trypsin. A tryptic digest of wild-type revealed a 48-kDa band by
SDS-PAGE, that was resistant to further digestion (Fig. 5). The 48-kDa peptide fragment was
missing both the amino-terminal 22 amino acids and the
carboxyl-terminal 145 amino acids of VLCAD. The molecular mass of the
nondenatured trypsinized VLCAD was 98 kDa, by gel filtration
chromatography, indicating that it is a homodimer of the 48-kDa
polypeptide. The FAD content was 2 mol/mol of dimer, the same as the
intact enzyme. Tryptic digestion of E422Q, E422D, F418T, and F418Y
yielded the same 48-kDa peptide as that of wild-type. On the other
hand, F418L (Fig. 5) and F418V (data not shown) were nearly completely
digested by trypsin as the 48-kDa band was barely visible. When
apo-VLCAD was digested with various amounts of trypsin, only a faint
48-kDa band was observed as with F418L and F418V (Fig. 5). This result
suggests that the lack of bound FAD is responsible for the increased
susceptibility of F418L and F418V to trypsinization.

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Fig. 5.
Tryptic cleavage of wild-type, mutant, and
apo-VLCADs. Three µg of protein was incubated with 0.03, 0.1, or
0.3 µg of trypsin in 20 µl of 50 mM potassium phosphate
(pH 7.5) at 37 °C for 30 min. The reaction was terminated by the
addition of phenylmethylsulfonyl fluoride (final concentration 1 mM) and then subjected to SDS-PAGE. The gel was stained
with Coomassie Brilliant Blue R-250.
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DISCUSSION |
Two subfamilies of acyl-CoA dehydrogenases may be distinguished by
the position of a catalytic glutamate residue: one subfamily corresponds to Glu-376 of MCAD, and the other corresponds to Glu-261 of
LCAD (12, 13, 15). Sequence alignments suggest that SCAD and VLCAD
belong to the MCAD subfamily (Glu-376), while IVD belongs to the LCAD
subfamily (Glu-261) (Fig. 1). Recently, Glu-368 of SCAD, which
corresponds to Glu-376 of MCAD, was confirmed to be a catalytic residue
(14). In the present study, we provide mutational evidence for Glu-422
of VLCAD being a catalytic residue.
Replacing Glu-422 of VLCAD with Gln caused a complete loss of enzyme
activity, while replacement with Asp decreased the
Vmax value to 10% of wild-type without a
significant change in the Km (Table II), suggesting
that the carboxylate of Glu-422 is intimately involved in the catalytic
function in VLCAD protein. The 
-dehydrogenation reaction
performed by acyl-CoA dehydrogenases involves the formation of a charge
transfer complex, which is initiated by the abstraction of the
-proton from acyl-CoA by the active site carboxylate, followed by
transfer of
-hydride to the N-5 position of enzyme-bound FAD (10,
11, 27). The charge transfer complex quenches the FAD absorbance at 450 nm and produces a new absorption band at 580 nm due to the disruption of the extended
-electron system of the FAD isoalloxazine ring (26-29). Measuring the UV visible spectra in the absence and the presence of palmitoyl-CoA demonstrated that E422Q was unable to form a
charge transfer complex in contrast to wild-type and other acyl-CoA
dehydrogenases which could form charge transfer complexes. Similarly,
only slight quenching of the absorbance at 450 nm was observed with
E422D. These data strongly suggest that Glu-422 of VLCAD is the
catalytic residue involved in the abstraction of the
-proton from
acyl-CoA.
A phylogenetic tree of human acyl-CoA dehydrogenases and acyl-CoA
oxidase, a peroxisomal enzyme which catalyzes the initial step of fatty
acid
-oxidation (30), was prepared (Fig.
6). Because the entire sequences of VLCAD
and acyl-CoA oxidase extend approximately 180 amino acids from the
carboxyl-terminal side of other acyl-CoA dehydrogenases, the regions
1-443 of VLCAD (9) and 1-441 of acyl-CoA oxidase (30), respectively,
were subjected to the phylogenetic analysis with the entire sequences
of other acyl-CoA dehydrogenases. The tree reveals that VLCAD and
acyl-CoA oxidase are separated from other acyl-CoA dehydrogenases and
that SCAD, MCAD, LCAD, and IVD are classified into two groups
consistent with the case of the position of catalytic glutamate. In
acyl-CoA oxidase, glutamate exists at the position corresponding to
Glu-376 of MCAD but not at the position corresponding to Glu-261 of
LCAD (30). Catalytic residue in the ancestor protein might be located at the position corresponding to Glu-376 of MCAD.

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Fig. 6.
Phylogenetic tree of human acyl-CoA
dehydrogenase family. The regions 1-443 of VLCAD (9), 1-441 of
acyl-CoA oxidase (30), and entire sequences of SCAD, MCAD, LCAD, and
IVD were analyzed by a MALIGN program at DDBJ homepage server.
AOX, acyl-CoA oxidase.
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F418L is a disease-causing mutation in human VLCAD
deficiency.2 In the present study, a large amount of the
F418L protein was successfully synthesized using the baculovirus
expression system. However, the cDNA-expressed F418L protein was
colorless because of the absence of enzyme-bound FAD (Table I and Fig.
3D) which led to a complete loss of catalytic activity
(Table II). Replacing Phe-418 with valine also caused the loss of
enzyme-bound FAD (Table I). These findings indicate that Phe-418 is
important for the binding of FAD to VLCAD. We prepared two additional
Phe-418 mutants, replacing phenylalanine with threonine and tyrosine,
which correspond to the residues in SCAD and MCAD, respectively (Fig.
1). F418T and F418Y bound as much FAD as wild-type, but had only 68 and 25% of the Vmax value of the wild-type enzyme,
respectively, without a significant change in the Km
toward palmitoyl-CoA and the electron transfer dye (Tables I and II).
Interestingly, the spectral data indicated that these two mutants could
form a charge transfer complex but with altered
max and
Kred values (Fig. 4). In MCAD-octanoyl-CoA
complex (13), the
-
bond of octanoyl-CoA is sandwiched between
the carboxylate of Glu-376 and the isoalloxazine ring, likely to allow
the electron transfer easily (Fig. 7).
Tyr-372 of MCAD, the residue corresponding to Phe-418 of VLCAD, is near the isoalloxazine ring, although it does not appear to be involved in
FAD binding. On the other hand, electron transfer flavoprotein, an
electron acceptor, is predicted to interact with acyl-CoA dehydrogenase through the sinister side of flavin to accept the electron from the
flavin ring (13). The replacement of Phe-418 with threonine and
tyrosine in VLCAD would be predicted to change the position of the
isoalloxazine ring in the complex formed between the catalytic residue
Glu-422, the
-
bond of acyl-CoA, and the site interacting with
the electron acceptor.

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Fig. 7.
Positions of Tyr-372 and Glu-376 in
three-dimensional structure of MCAD complexed with octanoyl-CoA.
The data was obtained from Brookhaven Protein Data Bank with accession
code 3MDE-A.
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Saijo and Tanaka (31) demonstrated that the isoalloxazine ring of FAD
is required for the formation of the core in the folding of MCAD
subunit into the tetramer assembly. Wild-type VLCAD contains a domain
that is resistant to trypsinization (Fig. 5) and extends from Lys-23 to
Arg-470 of mature VLCAD (which includes residues Phe-418 and Glu-422).
This 48-kDa peptide corresponds to the highly homologous regions of
other acyl-CoA dehydrogenases. Trypsinization of VLCAD produced a
48-kDa homodimer that contained the same amount of FAD as native VLCAD.
These findings suggest that the VLCAD subunit tightly binds between
amino acids 23 and 470 and that this region includes the catalytic
domain. On the other hand, the FAD-defecient VLCADs (F418L, F418V, and
apo-VLCAD) were capable of forming dimers, but showed increased
sensitivity to trypsinization. Some degree of folding change may occur
by the lack of FAD.