The Y42H mutation in medium-chain acyl-CoA dehydrogenase,
which is prevalent in babies identified by MS/MS-based newborn
screening, is temperature sensitive
Linda O’Reilly
1
, Peter Bross
2
, Thomas J. Corydon
3
, Simon E. Olpin
4
, Jakob Hansen
2
, John M. Kenney
5,6
,
Shawn E. McCandless
7
, Dianne M. Frazier
8
, Vibeke Winter
2
, Niels Gregersen
2
, Paul C. Engel
1
and Brage Storstein Andresen
2,3
1
Department of Biochemistry and the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland;
2

levels of the Y42H variant in comparison to wild-type were
decreased at higher temperature though to a lesser extent
than for t he K304E variant. To d istinguish between effects of
temperature on folding/assembly and t he stability o f the
native enzyme, the thermal stability of the variant proteins
was studied after expression and purification by dye affinity
chromatography. T his s howed that, compared w ith t he wild-
type e nzyme, the thermostability of the Y42H variant was
decreased, but not to the same degree as that of the K304E
variant. Substrate binding, i nteraction with the natural
electron acceptor, and the binding of the prosthetic group,
FAD, were only slightly affected by the Y42H mutation. Our
study suggests that Y42H is a temperature sensitive muta-
tion, which is mild at low temperatures, but may have
deleterious effects at increased temperatures.
Keywords: chaperones; newborn screening; protein folding;
thermostability.
Medium-chain acyl-CoA dehydrogenase (MCAD)
(EC 1 .3.99.3) is a homotetrameric enzyme that catalyses
the initial oxidation step in the b-oxidation of medium-
chain fatty acids in mitochondria [1]. Medium-chain acyl-
CoA dehydrogenase deficiency (MCADD; MIM 201450) is
the commonest fatty acid o xidation defect occurring in
Europe, affecting Caucasians o f North-western European
origin, with an incidence as high as 1 : 8000 live births [2].
Symptoms can be quite broad, ranging from hypoglycaemia
and lethargy to seizures, coma and s udden death. Some
genetically predisposed patients remain asymptomatic
throughout life [3–5]. The disease can present at any time
of life, from the neonatal period [6,7] to adulthood [8–10].

folding of t he polypeptide into monomer is facilitated by the
Hsp60 chaperonin system and the t etramer i s formed [18].
We and others have previously reported that the K304E
mutation influences this biogenesis process at several steps
by affecting the folding of the monomer, impairing
oligomerization and destabilizing the tetramer [18–23].
The folding/tetramerization defect of the K304E mutant
protein can be partly overcome by increasing the amount of
chaperonins or by lowering the culture temperature when
the recombinant protein is expressed in Escherichia coli
[21,22]. Similarly, many of the other disease-causing muta-
tions in MCAD that have been characterized seem to
influence folding and can be rescued to a varying extent by
chaperonin co-overexpression and/or lowering the growth
temperature [5,11]. Recently a new prevalent mutation
199TfiC, causing the missense mutation Y 42H, w as
identified [11,24]. It was found in newborns heterozygous
for the prevalent 985AfiG mutation who showed an
abnormal acylcarnitine profile in an MS/MS screen of
blood spots, and the carrier frequency in the US was
determined to be 1/500 [11]. The carrier frequency of the
985AfiG (K304E) mutation in t he same area ranges from
1/80 to 1/100, making Y42H the second most prevalent
mutation of MCAD deficiency [ 11]. Y42H has also been
found to b e present in Germany, New South Wales and
Spain ([24] and B . S . Andresen & N. G regersen, unpub-
lished results). Despite the fact that the Y42H mutation is so
prevalent, and gives rise to an abnormal acylcarnitine profile
in blood spots, it has so far not been reported in clinically
manifesting patients [11,24]. Therefore, the clinical implica-

equilibration buffer for 1 column vol., then 4–5 column vols
of 20 m
M
KPi, 50 m
M
KCl pH 7.2, until no m ore
contaminants eluted. The enzyme was then e luted with
20 m
M
KPi, 200 m
M
KCl pH 7.2. The eluate was concen-
trated and d esalted utilizing an A micon Centricon device
(M
r
cutoff 30 kDa). The protein was loaded onto a 20-mL
Procion red HE-3B dye affinity column (Procion dyes were
a g enerous gift from C. V. Stead of the former Imperial
Chemical Industries, Dyestuffs Division, Blackley, Man-
chester, UK), linked t o Sepharose (Pharmacia Biotech), pre-
equilibrated w ith 5 0 m
M
KPi, 50 m
M
KCl p H 7 .2 (1 cm
diameter) The column was washed with th e pre-equilibra-
tion buffer, until no more contaminants eluted. The enzyme
was then eluted by adding 1 mL of 3.5 m
M
of the substrate

in the presence of [
35
S]methionine (20 lCi per 50 lL
reaction, 10 lCiÆlL
)1
; Amersham Bio sciences) using the
TnT co upled reticulocyte lysate kit (Promega) according to
the manufacturer’s protocol. The translation was stopped
by the addition of cycloheximide (0.15 lgÆmL
)1
final
concentration). Rat liver mitochondria were isolated as
described p revio usly [28,29]. The translation p roduct was
mixed w ith i solated m itochondria and imported into
mitochondria essentially as d escribed previously [25]. The
mixture was then incubated at 26 °Cor41°C, and
intramitochondrial biogenesis was followed by withdrawing
aliquots at different time points (0–180 min). Samples were
treated as described previously [25]. The supernatant
fraction, which contained s oluble matrix components,
including MCAD enzyme protein and complexes thereof,
was analysed by native (nondenaturing) PAGE (4–15%
Tris/HCl Criterion gels from Bio-Rad) and by SDS/PAGE
(12.5% Tris/HCl Criterion gels from Bio-Rad) as described
previously [25]. The pellet fraction, which contains insoluble
MCAD protein, was analysed by SDS/PAGE. Radio-
labelled MCAD protein was v isualized by phosphor
imaging using an Amersham Biosciences Phosphorimager
4054 L. O’Reilly et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(STORM 840) and the bands were quantified using

ning and Olpin [31,32]. Patient fibroblasts, along with
controls, were seeded into 24-well plates and incubated at
34 °C, 37 °Cand39°C for 72 h p rior to assay at these
temperatures.
Structure analysis of MCAD by synchrotron radiation CD
(SRCD)
CD studies were preformed using the UV 1 beamline at the
Institute for Storage Ring Facilities at the University of
Aarhus. Samples were prepared in 20 m
M
KPi pH 7.2
buffer. Samples at concentrations of 330 lgÆmL
)1
and
50 lgÆmL
)1
and buffer (for b aseline correction) were
placed in a 0.5-mm light path Suprasil quartz cell (Helma)
for CD spectroscopy. CD spectroscopy of all the samples
were made under the same conditions as a function of
temperature (at fixed points between 30 °Cand75°C)
and time (5 min equilibration at each temperature). The
baseline (buffer only) spectra were recorded before and
after t he CD scan of each sample using the same cell as
that u sed for the sample and under the s ame c onditions
(specifically temperature). Both the baselines and protein
scansweremadeinduplicateandthemeanbaseline
subtracted from the mean scan, before plotting. The
spectra of the baseline-corrected 50-lgÆmL
)1

somewhat lower than the previously published value of
12 l
M
[33,34]. We found that the Y42H mutant protein has
approximately the same maximum velocity (V
max
¼
24.2 ± 0.7 · 10
3
nmol ferricenium/mgÆmin
)1
) as the wild-
type enzyme (V
max
¼ 24.6 ± 0 .6 · 10
3
), but the Y42H
protein has a higher K
m
than wild-type (5.2 ± 0.5 l
M
),
indicating that the substrate binding is slightly impaired.
The maximum velocity of the K304E mutant protein
was only one third of the wild-type value (V
max
¼ 8.2 ±
0.3 · 10
3
).

growth temperature [11], we decided to use this eukaryotic
system [25] to investigate the influence of t emperature on
biogenesis and s tability o f t he Y42H MCAD , c ompared
to wild-t ype a nd K3 04E MCAD. We p erformed in vitro
transcription/translation of the MCAD variants, imported
the products into purified rat liver mitochondria, and
monitored the time course of folding and formation/
stability of the tetramer. The studies were performed at
26 °Cand41°C (Fig. 1). At 26 °C the amounts of tetra-
mer formed increased until 120 min. T here is an obvious
difference between the amounts of K304E tetramers being
formed compared to wild-type, whereas the rate of tetramer
formation is only slightly decreased for the Y42H mutant
protein. Considering the fraction of soluble MCAD protein
that represents tetrameric enzyme, it appears that relatively
less Y42H tetramer is formed compared to wild-type. This
could be interpreted as indicating that folding of the
monomers into an assembly-competent conformation is
slowed for the Y42H protein, and/or that formation of
Ó FEBS 2004 The MCAD Y42H mutation is temperature sensitive (Eur. J. Biochem. 271) 4055
tetramers from the assembly competent monomers is also
slightly decreased. The observation that the amount of
soluble nontetrameric K304E protein increases over time,
and that it makes up a much bigger fraction at the last time
points than observed for the wild-type protein is consistent
with previous studies showing that the K304E protein has a
defect both in monomer folding and tetramer assembly [19].
In the studies performed at 41 °C (Fig. 1) it can be seen
that for the wild-type the amount of soluble protein reached
a peak within the first 10–30 min and the amount of

Because the experiments described above could not unam-
biguously delineate if the temperature sensitivity of the
Y42H mutant protein is caused by decreased thermostabi-
lity and/or biogenesis, we investigated the thermal stability
by generating thermostability curves with the purified
recombinant MCAD proteins. Preliminary thermal inacti-
vation profiles of crude extracts from E. coli cells over-
expressing Y42H or K304E mutant proteins, respectively,
have previously demonstrated that the thermal inactivation
profiles of K304E and Y42H are shifted to lower temper-
atures [11,21]. In the present study the residual enzyme
activity levels were measured at two MCAD pro-
tein concentrations (3.3 lgÆmL
)1
and 50 lgÆmL
)1
). At
3.3 lgÆmL
)1
a d ifference i s observed between the variant
proteins (Fig. 2A), with Y42H showing a decreased stability
at temperatures above 42 °C c ompared to w ild-type, and
K304E showing a more pronounced decrease. At the higher
protein concentration of 50 lgÆmL
)1
there is little difference
observed i n t he ther mostability b etween the various pro-
teins. Interestingly, all MCAD variant proteins show an
increased thermal stability at the higher concentration and a
further elevation of the concentration ( 0.33 mgÆmL

ABC
D
E
Fig. 2. Enzyme activity assays of wild-type, Y42H and K304E mutant protein at 3.3 lgÆmL
)1
and 50 lgÆmL
)1
for without BSA (A) and with
1mgÆmL
)1
BSA (C). The activity at the higher concentration o f 0.33 mgÆmL
)1
(B) is also shown. Note that the graphs for Y42H and the wild-type
mutant proteins are completely overlapping at this high p rotein concentration. The amount of protein, corresponding to the activity at 3.3 lgÆmL
)1
is seen by Western blot analysis of ( D) native ge l showing tetramer formation and (E) showin g the ratio of soluble (S ¼ supernatant fraction) and
insoluble (P ¼ pellet fraction) prote in by SDS/PAG E. Error bars ind icate the standard d eviation of the me an result.
Ó FEBS 2004 The MCAD Y42H mutation is temperature sensitive (Eur. J. Biochem. 271) 4057
tetramer falls together with the MCAD protein be coming
insoluble and aggregating.
SRCD of wild-type and mutant MCAD proteins
To determine whether the K304E and Y42H mutations
have an effect on the secondary structure of MCAD, the
purified variant proteins were analysed by SRCD [36],
which is more sensitive than conventional CD spectroscopy
at low wavelengths (< 190 nm) [37,38]. Analysis of the
three variant MCAD proteins at a protein concentration of
330 lgÆmL
)1
at 35 °C showed no significant difference in

in human cells
To investigate the relevance of the results obtained in the
model systems descr ibed a bove, we analysed steady-state
amounts of Y42H and K304E MCAD in immortalized
lymphoblastoid cells cultured at 34 °C, 37 °Cand39°Cby
Western blotting. Cells homozygous or heterozygous for the
K304E mutation, cells compound heterozygous for
the K304E and Y42H mutations and cells homozygous
for the wild-type allele were used (Fig. 4). At 34 °Cthereis
little difference between the amount of either tetramer or
soluble protein present in the K304E/wild -type heterozy-
gote, compared t o the K304E/Y42H heterozygote, indica-
ting that at this temperature there is little difference between
wild-type and the Y42H variant. However, if the tempera-
ture is raised to 37 °Cand39°C, the difference becomes
more obvious with much less MCAD protein present for the
K304E/Y42H heterozygote. In fact, both the levels of
soluble MCAD protein present in the SDS gel and the
amounts of MCAD tetramers present in the native gels
from the K304E/Y42H heterozygote a re comparable to
those observed from the K304E homozygote at 39 °C.
The effect of temperature on the mutant and wild-type
proteins was investigated further by measuring the
b-o xidation in fibroblast cells using myristic acid as
substrate. The results are shown in Fig. 5. As expected,
the K304E homozygote cells had the lowest activity level.
However, this level remains relatively unaffected by the
increasing tempe rature. The wild-type/K304E heterozygote
cells showed the highest activity level, and again were
relatively stable with increasing temperature. However, the

and might suggest that Y42H rarely precipitates clinically
manifested disease, and therefore could be regarded as a
ÔbenignÕ variant. Alternatively, patients with this mutation
may not be recognized because they exhibit a different
clinical presentation. Given the widespread use of MS/MS-
based newborn screening, it i s a cause for major c oncern
that this method ma y detect Ôbenign Õ variants of unknown
clinical significance, creating unwarranted anxie ty in parents
and health care professionals [39]. It is therefore of utmost
importance that t he consequences of the Y 42H mutation
are t horoughly i nvestigated, to distinguish b etween a
ÔbenignÕ MCAD variant that causes an abnormal acylcar-
nitine pattern, but is unlikely to cause disease, and a Ôdisease-
causingÕ MCAD variant. Therefore we have in t he present
study investigated how the Y42H mutation affected the
MCAD protein using studies in both in vitro systems and
patient cells.
Our investigation of purified recombinant protein showed
that the Y42H mutation only had a minimal effect on the
catalytic activity of the enzyme, the prosthetic group
binding, or interaction with the natural electron acceptor.
Together these d ata show that the Y42H mutation
compromises the enzymatic function to a minor degree. It
is unlikely that these changes alone could explain the
biochemical abnormality observed in newborns with the
Y42H muta tion.
Instead is seems that the biogenesis and/or stability of the
Y42H mutant enzyme is more significantly affected. This
was also indicated in previous experiments as overexpres-
sion in E. coli revealed that the temperature at which the

wild-type, but when the temperature was increased from
31 °Cto37°C, this activity was significantly decreased
(35–40% of wild-type). This impact of culture temperature
on enzyme activity levels for the Y42H mutant protein
could indicate that it is a Ôfolding mutantÕ, like many of the
previously characterized disease-causing mutations in
MCAD [5,21,23]. However, unlike t he MCAD proteins
with folding mutations, overexpression of chaperonins
appeared to have very little o r no e ffect on the Y42H
protein [11]. A thermal stability curve of crude lysates from
E. coli overexpressing mutant or wild-type M CAD indica-
ted a dec rease in the thermal stability of Y42H protein as
compared to the wild-type suggesting that the temperature
effect might be due to a decreased s tability o f the active
enzyme once it has acquired the native structure [11].
In the present study we investigated the biogenesis/
stability of the Y42H mutant protein further and c ompared
it to wild-type and K304E mutant MCAD. Our results with
the coupled in vitro transcription/translation of MCAD
proteins followed by import into rat liver mitochondria
corroborated previous studies of the K304E mutant protein
[19], showing that this mutation has a drastic effect on
formation of t etrameric MCAD protein, probably as a
result of a combined defect in the folding of monomers and
in assembly/stability of the tetramer. It was clear from the
present studies that the amounts of tetramers formed for the
Y42H mutant variant were slightly decreased compared to
wild-type and the effect was exacerbated at increased
temperature.
The in vitro translation studies clearly demonstrated that

placed on the MCAD tetramer, i.e. by increasing the
temperature, the t etramer has an increased tendency to
dissociate into dimers and monomers. At low MCAD
concentrations, the probability of an MCAD monomer/
dimer meeting other monomers/dimers and re-forming the
tetramer is lower than at h igh MCAD concentration. This
would explain the increased thermal s tability observed at
high MCAD concentrations. The effect of the Y42H
mutation may thus be primarily on stability of the native
structure resulting in both temperature and concentration
sensitivity.
From the crystal structure [40] it can be seen that
tyrosine-42 is placed in the small helix B with the side chain
pointing to the surface of the tetramer (Fig. 6). The
aromatic ring of tyrosine-42 is packed between residues
and this structure appears to be part of an i nteraction
network that stabilizes the fold of helices A, B and C and
links it to the e dge of the b-sheet domain. Substitution of
tyrosine-42 with histidine may be expected to disturb these
interactions. The side chain of histidine is somewhat smaller
than that of tyrosine and hydrophobic interaction s of
carbon atoms in the aromatic ring of tyrosine with the
neighbouring residues would b e altered or abolished. This
could result in loosening of th e stability of the structure in
this part of the monomer. Although tyrosine-42 is distant
from subunit interaction interfaces, increased breathing of
the helix A, B, C fold and its anchoring to the b-sheet
domain could result in an increased tendency of the tetramer
to dissociate. At high MCAD concentrations and low
temperature, reassociation would dominate, whereas at high

was almost undetectable at 39 °C. Y42H also showed the
greatest reduction in the enzyme activity level. The reason
for the temperature sensitivity of the Y42H and K304E to
become so pronounced in fibroblasts and lymphoblasts is
most probably that t he concentration of t he endogenous
MCAD proteins in these cells are many fold l ower than
those i nvestigated in t he experiments p erformed with
purified enzymes. The t emperature sensitivity of the
K304E and Y42H proteins was also r eflected a s decre ases
in b-oxidation activities of cultured fibroblasts with increas-
ing temperatures, indicating that this effect is observable in
intact cells. Interestingly, the activity of the Y42H/K304E
heterozygote approached that of a K304E homozygote
demonstrating that at increased temperature the Y42H/
K304E genotype can result in b-oxidation levels dropping
almost to the levels o bserved in p atients with clinically
manifested disease. These observations clearly show that
the steady-state amounts of functional MCAD enzyme in
human cells compound heterozygous for the Y42H and
K304E mutations is highly dependent on temperature.
In conclusion our results show that Y42H is indeed a mild
mutation, but that its effect becomes more pronounced at
higher temperatures. These data suggest that individuals
with the Y42H/K304E genotype a re likely to experience a
further lowering of t heir MCAD enzyme activity in relation
to increased body temperature as may be experienced
during intercurrent infection. It is not easy to judge if this
will lead to c linical symptoms as a result of metabolic
decompensation, but several individuals who are compound
heterozygotes for the Y42H and K304E mutations, identi-

manifestation. Until m ore knowledge i s gained, these
individuals should be considered as being at risk of disease
manifestation.
Acknowledgements
We are grateful to Bridget Wilcken for c ontributing patient fibroblasts.
We thank Linda Steinkrauss and Charles Stan ley for sharing clinical
information. We thank Christian Knudsen f or careful culturing of
patient cells. We are grateful to the Institute for Storage Ring Facilities
(ISA), University of Aarhus, for a ccess to the CD facility on the UV1
beamline at ASTRID, and especially for the help provided by Søren
Vorrening. This wo rk was sup ported by grants from the Enterprise
Ireland International Collaboration Programme, the Faculty of
Science, University College Dublin (through the award of a demonst-
ratorship), the Danish Medical ResearchCouncilandtheMarchof
Dimes (1-FY2003-688).
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