Molecular defect of isovaleryl-CoA dehydrogenase in the
skunk mutant of silkworm, Bombyx mori
Kei Urano
1
, Takaaki Daimon
1
, Yutaka Banno
2
, Kazuei Mita
3
, Tohru Terada
4
, Kentaro Shimizu
4,5
,
Susumu Katsuma
1
and Toru Shimada
1,4
1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
2 Institute of Genetic Resources, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
3 Division of Insect Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
4 Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
5 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
Introduction
Isovaleryl-CoA dehydrogenase (IVD; EC 1.3.99.10) is
a tetrameric, mitochondrial flavoenzyme that catalyses
the third step of leucine degradation in which isovale-
ryl-CoA is converted to 3-methylcrotonyl-CoA. IVD is
a member of the acyl-CoA dehydrogenase (ACAD)
family of enzymes, all of which share significant

were constructed with a baculovirus expression system and the subsequent
enzyme activity of sku-type BmIVD was shown to be significantly reduced
compared with that of wild-type BmIVD. Molecular modelling suggested
that this reduction in the enzyme activity may be due to negative effects of
G376V mutation on FAD-binding or on monomer–monomer interactions.
These observations strongly suggest that BmIVD is responsible for the sku
locus and that the molecular defect in BmIVD causes the characteristic
smell and prepupal lethality of the sku mutant. To our knowledge, this is,
aside from humans, the first characterization of IVD deficiency in metazoa.
Considering that IVD acts in the third step of leucine degradation and the
sku mutant accumulates branched-chain amino acids in haemolymph, this
mutant may be useful in the investigation of unique branched-chain amino
acid catabolism in insects.
Abbreviations
ACAD, acyl-CoA dehydrogenase BmIVD, Bombyx mori isovaleryl-CoA dehydrogenase; EST, expressed sequence tag; IVD, isovaleryl-CoA
dehydrogenase; PMS, phenazinemethosulfate; SNP, single nucleotide polymorphism.
4452 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS
IVD dysfunction is well characterized in humans as
the first recognized organic acidaemia (isovaleric acida-
emia), which causes recurrent episodes of vomiting,
lethargy, developmental delay and sometimes acute
neonatal death [2,3]. Isovaleric acid, one of the deriva-
tives of isovaleryl-CoA, is abnormally excreted in
blood and causes a characteristic sweaty feet odour in
patients.
The isovaleric acid-emanating silkworm mutant
skunk (sku) was first described over 30 years ago as an
‘odorous silkworm’ [4,5]. The sku gene is an autosomal
recessive lethal gene and the sku mutant exhibits
prepupal lethality (Fig. 1). However, the gene responsi-

Identification of the B. mori isovaleryl-CoA
dehydrogenase (BmIVD) gene as a candidate for
the sku mutant
A search of the silkworm expressed sequence tag
(EST) database revealed the existence of several puta-
tive acyl-CoA dehydrogenase genes in silkworm.
Among them, one EST clone, fdpeP14_F_F20, exhib-
ited the highest homology (69%) to human IVD. After
analysis of the full-length sequence of this EST clone,
it was apparent that two key residues distinguishing
IVD from other ACAD family members are both con-
served. One is the catalytic base E254, which abstracts
sku / +
sku
sku / sku sku / +
sku
sku / sku
B A
Fig. 1. Phenotypes of control silkworm
(sku ⁄ +
sku
) and the skunk mutant (sku ⁄ sku)
at (A) day 2 of fifth instar and (B) 10 days
after spinning. Until spinning, the skunk
mutant larva develops normally (A). After
spinning, the mutant dies without pupation,
whereas control larva successfully moults to
pupa (B). Scale bar, 10 mm.
K. Urano et al. Odorous silkworm mutant
FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4453

2.3
2.0
1.1
(kb)
B
C
A
wt
sku
Skunk-RT2 gPCRsku-R2
T
poly(A)
Start codon Stop codon
Probe
skunk-RT1
+1 +1127 +1251 +1300–37
G
poly(A)
(nt)
rp49
BmIVD
*
G376V
Fig. 2. Cloning of the BmIVD gene. (A) RT-
PCR analysis revealed the BmIVD band
amplified from whole-body RNA of standard
strains p50T and c108T as well as the sku
mutant. Migrations of the molecular mass
marker and control gene rp49 are indicated.
(B) Full-length mRNA of wild-type and sku

for valine (G376V) in BmIVD. An investigation of the
nucleotide sequence at the 1127th position in eight
+
sku
⁄ +
sku
silkworm strains revealed that all strains
conserved the canonical guanine at this site (data not
shown). To examine other possible variations, genome
sequences of BmIVD were determined and 11 SNPs
between BmIVDs of wild-type and sku were found, in
addition to 1127G>T. However, all were located on
the introns of BmIVD (data not shown). Thus, these
11 SNPs appeared to have no functional influence on
this gene.
Alignment of the amino acid sequences of IVDs
(Fig. 2C) exhibited that BmIVD is highly homologous
to other IVDs throughout the entire region. Notably,
the glycine residue corresponding to G376 of BmIVD,
which was substituted to valine in the sku mutant, is
highly conserved from bacterial to mammalian IVDs,
indicating the importance of this residue.
Linkage analysis between sku and BmIVD genes
To determine the consistency between the sku pheno-
type and the BmIVD genotype, linkage analysis
between the wild-type and sku strains was performed.
For this, crossing of the strain a85, in which sku locus
is marked with or, a recessive ‘oily’ gene that causes
translucent epidermis, was performed. As represented
in Fig. 3, four genotypes from F

that BmIVD is expressed in various tissues, ranging
from digestive organs such as midgut to reproductive
organs such as ovary and testis or the respiratory
organ trachea (Fig. 4B). Among these tissues, fat body
and midgut showed higher expression levels than other
tissues. It is noteworthy that both tissues play essential
roles in nutrient turnover in insects. Namely, nutrients
are digested and absorbed in the midgut and stored
and metabolized in the fat body which is equivalent to
liver in mammals. Thus, it is likely that BmIVD may
G/G G/T T/T
1127
|
P
F
1
1127
|
1127
|
or +
++
or sku
++
or +
or sku
or sku
or sku
or sku
or +

1127th base pair
of BmIVD ORF
G ⁄ GG⁄ TT⁄ T
Normal (+ ⁄ +, sku ⁄ +)30 12180
Oily and nonodorous (sku ⁄ +) 60 0 60 0
Oily and odorous (sku ⁄ sku)43 0 043
K. Urano et al. Odorous silkworm mutant
FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS 4455
participate in amino acid catabolism for energy pro-
duction in the silkworm.
Expression of recombinant BmIVD by the
baculovirus expression system
To evaluate the effect of the G376V amino acid substi-
tution in the sku mutant on the IVD activity and sub-
strate specificity, we first carried out overexpression
and purification of wild-type and sku-type (G376V)
BmIVDs. Because the expression level in IVD-recom-
binant Escherichia coli was previously reported to be
extremely low and 5¢-end alteration to mimic codon
usage of E. coli is necessary for improved expression
levels [12], a baculovirus expression system was
employed to overexpress the BmIVD. Sf9 cells were
infected with a recombinant baculovirus that expresses
the full-length BmIVD with His-tagged sequences at
the C-terminus. As indicated in Fig. 5A, the expression
level of recombinant BmIVD was sufficiently high that
a putative BmIVD band could be observed in Coomas-
sie Brilliant Blue staining. Western blot analysis
revealed that the molecular mass of the expressed
BmIVD is apparently lower than that of the predicted

Next, sku-type BmIVD (G376V) was examined to
determine if it retained enzymatic activities. As indicated
in Fig. 6, sku-type BmIVD exhibited only faint ACAD
activities against all the substrates investigated. This
1.3 kb
FB MG MT EP
wt
FB MG MT EP
sku
Actin3
BmIVD
rp49
BmIVD
B
A
Fig. 4. Expression profiles of BmIVD. (A) Northern blotting compares the expression levels of BmIVD from several tissues obtained from
fifth instar larvae at day 2 of wild-type (p50T) and mutant (sku) strains. Total RNA (5 lg) prepared from fat body (FB), midgut (MG), Malpi-
ghian tubule (MT) and epidermis (EP) were blotted and hybridized with the digoxigenin (DIG)-labelled probe. The arrowhead indicates the
positive signal. Silkworm Actin3 is represented as a control. (B) RT-PCR analysis using cDNAs from 15 tissues of wild-type p50T strain are
indicated. Lane 1, brain (BR); lane 2, prothoracic gland (PG); lane 3, salivary gland (SaG); lane 4, central nervous system (CNS); lane 5, tra-
chea (TR); lane 6, fat body (FB); lane 7, ovary (OV); lane 8, testis (TES); lane 9, anterior silk gland (ASG); lane 10, middle silk gland (MSG);
lane 11, posterior silk gland (PSG); lane 12, midgut (MG); lane 13, hindgut (HG); lane 14, Malpighian tubule (MT); lane 15, epidermis (EP).
Silkworm rp49 was the control.
Odorous silkworm mutant K. Urano et al.
4456 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS
result demonstrates that the point mutation at Gly376
in BmIVD almost totally disrupts the function of
BmIVD as an enzyme, indicating BmIVD dysfunction
in sku mutants.
Sequence alignment and position of the

˚
in the mutant structure
(Fig. 7B). To avoid these overlaps, the mutant proba-
bly has a different structure in this region.
Discussion
In this study, a candidate gene approach was utilized
to discover the gene responsible for the odorous silk-
worm mutant sku. The candidate gene BmIVD was
identified and a single nucleotide substitution was
found in the codon of a highly conserved residue, not
only in the species, but also in all enzyme family mem-
bers (Figs 2C and 7A). It was demonstrated that this
substitution perfectly cosegregated with the sku loci
(Fig. 3 and Table 1) and dramatically decreased the
enzymatic activity (Fig. 6). These genetic and biochem-
ical data, along with previous observations that the
sku mutant accumulates isovaleric acid and branched-
chain amino acids, strongly indicate that a single
amino acid substitution (G376V) in BmIVD is respon-
sible for the sku mutant. In the sku mutant, dysfunc-
tion of BmIVD would cause hydrolytic degradation of
75
50
37
25
(kDa)
1 2 3 4 5 6 7 8 9 10
AB
C
D

that Gly376 (corresponding to position 354 in human
IVD) is essential for common mechanisms in ACADs.
ACAD is a homotetrameric or homodimeric flavopro-
tein with each monomer containing one molecule of
FAD [17]. FAD not only serves as a catalyst, but also
bridges the monomers; it is located at the interface
between the monomers and forms many hydrogen
bonds with both [16,18]. Molecular modelling revealed
that the G376V mutation causes the side chain of
Val376 to overlap with other residues, possibly result-
ing in changes in the loop structure. Although the
loop has only been characterized to make a hydrogen
bond with FAD [16], the model structures indicated
that it is also involved in interactions with the neigh-
bouring monomer. As shown in Fig. 7B, Tyr377 and
Asn379 in the loop region interact with Leu236 and
Asp234, respectively, of the neighbouring monomer
via a hydrogen bond. These results suggest that the
G376V mutation alters the structure of the loop
region and affects the interactions between monomers
and with FAD. Imperfect FAD-binding or tetramer
formation would lead to disappearance of the enzy-
matic activity (Fig. 6). Recent clinical mutation stud-
ies about ACAD deficiency in humans support this
prediction [17]. An identical substitution at the
homologous position (G371V) in human short-chain
acyl-CoA dehydrogenase has also been reported and,
though this protein’s enzymatic activity was not men-
tioned, in vitro import studies revealed that this muta-
tion led to a temperature-dependent inability to form

at levels  4 times higher in females and  7–12 times
wt
120
100
80
60
40
20
0
sku
Relative activity (%)
*
*
**
IV-CoA IB-CoA HX-CoA
Fig. 6. Relative enzymatic activities of wild-type (wt) and sku-type
BmIVD (sku). Enzymatic activities of isovaleryl-CoA (IV-CoA), isobu-
tyryl-CoA (IB-CoA) and hexanoyl-CoA (HX-CoA) were assayed by
the 2,6-dichloroindophenol ⁄ PMS dye-reduction method. The data
show means ± SD of pooled data from two independent experi-
ments each performed in triplicate. (**P < 0.0001, *P < 0.05, one-
tailed, Student’s t-test).
Odorous silkworm mutant K. Urano et al.
4458 FEBS Journal 277 (2010) 4452–4463 ª 2010 The Authors Journal compilation ª 2010 FEBS
higher in males than in normal silkworms [6]. In human
cases, however, patients with isovaleric acidaemia do
not exhibit such accumulation of amino acids [2]
because an enzyme reaction one step before IVD dehy-
drogenation, in which a-ketoisocaproate is catalysed by
branched-chain a-keto acid dehydrogenase, is irrevers-

A
G376V
B
G376
V376 I369
I369
BmIVD
IVD
IBD
SBCAD
GCD
SCAD
MCAD
LCAD
VLCAD
ACAD9
ACAD10
398
376
399
415
415
369
377
413
423
427
938
755
J

The silkworms were reared on fresh mulberry leaves in an
insect rearing chamber under standard conditions (25 °C,
12L : 12D photoperiod). Sf9 cells were cultured at 27 °Cin
TC-100 insect medium (SAFC Biosciences, Lenexa, KS,
USA) supplemented with 10% fetal bovine serum. Autogra-
pha californica multiple nucleopolyhedrovirus (AcMNPV)
was propagated in the Sf9 cells as described previously [25].
All tissues and cells to be examined were washed twice in
NaCl ⁄ P
i
(137 mm NaCl, 2.7 mm KCl, 8.1 mm Na
2
HPO
4
,
1.5 mm KH
2
PO
4
), immediately frozen in liquid nitrogen
and stored at )80 °C. PCRs were performed using the
ExTaq Kit (Takara Bio, Shiga, Japan), unless otherwise
mentioned.
Isolation of B. mori cDNA encoding the IVD-like
gene
To identify the Bombyx gene which is homologous to the
IVD gene, the EST database was screened [26]. The cDNA
clone fdpeP14_F_F20 exhibited the highest homology to
human IVD and was subjected to further analysis. Assess-
ment of the genetic loci of the EST clone was performed

described above.
Preparation and sequencing of the BmIVD
genomic clone
Genomic DNA was extracted from the silk glands of fifth
instar larvae according to standard methods [27]. Because
the genomic structure of BmIVD is long ( 11 kb), the gen-
ome sequence was divided into two parts and sequenced
separately. The genomic sequence of BmIVD was PCR-
amplified using the TaKaRa LA Taq Kit (Takara Bio).
Amplified PCR fragments were subcloned and sequenced as
described above. Full-length cDNA and genomic sequences
of wild-type BmIVD were deposited into the GenBank ⁄
EMBL ⁄ DDBJ data bank with accession numbers
AB458683 for cDNA and AB462483 for genomic DNA.
Linkage analysis between sku and BmIVD
The heterozygous mutant of sku can be identified using the
sku-linked recessive oily gene or. Crossing was performed
as indicated in Fig. 3. Thirty normal larvae, 60 oily but
nonodorous larvae and 43 oily and odorous larvae were
screened at fifth instar. To extract genomic DNA, caudal
portions of the larvae were cut and homogenized with a
pestle and DNeasy Blood and Tissue Kit (Qiagen, Venlo,
The Netherlands) was used. The genomic DNA was ampli-
fied by PCR with primers PCRseqF and gPCRsku-R1,
which were designed to amplify the fragment that contains
the substitution site in BmIVD. The PCR product was then
cleaned using the QIAquick PCR Purification Kit (Qiagen)
and directly sequenced as described above.
Northern blot analysis
Total RNA from the fat body, midgut, Malpighian tubule

ium was discarded and cells were suspended in 10 mL
NaCl ⁄ P
i
. The suspension was centrifuged at 3000 g for
10 min and the cell pellet was washed and stored at )80 °C
until use. To purify recombinant proteins, harvested cells
were resuspended in 5 mL of 50 mm potassium phosphate
buffer (pH 8.0) per dish, together with protease inhibitor
cocktail tablets (Roche). The cells were lysed by sonication
for 1 min in the Branson Sonifier 250 (Branson, Danbury,
CT, USA) and added with the same volume of binding buffer
(5 mm imidazole, 20 mm sodium phosphate, 500 mm NaCl,
pH 7.4). After centrifugation at 14 000 g for 10 min, the
resulting supernatants were loaded onto a HisGraviTrap col-
umn (GE Healthcare Bioscience, Little Chalfont, UK). The
eluate was dialysed twice in 100 mm potassium phosphate
(pH 8.0) and 100 mm NaCl using the Slide-A-Lyser Dialysis
Cassette (Pierce, Rockford, IL, USA). The protein concentra-
tion was determined using the Coomassie Plus Protein Assay
Reagent (Pierce) with bovine serum albumin as the standard.
Expression and purification of recombinant protein was
confirmed by SDS ⁄ PAGE [30] and western blot as
described previously [31].
Enzyme assays
The isovaleryl-CoA dehydrogenase activity was assayed
spectrophotometrically by the dye-reduction method using
2,6-dichloroindophenol as an electron acceptor and phenaz-
inemethosulfate (PMS) as an intermediate electron carrier
as described previously [32,33], with slight modifications.
The incubation medium was composed of 50 mm potassium

erate the models [35]. Conformations of the side chains
were refined with SCWRL 3.0 [36] and the quality of the
models was evaluated with Verify3D [37].
Acknowledgements
This work was supported by grants from MEXT (Nos.
17018007 to T.S.), JSPS (21248006 to TD and TS),
MAFF-NIAS (Agrigenome Research Program) and
JST (Professional Program for Agricultural Bioinfor-
matics), Japan. The silkworm strains and DNA clones
were provided by the National Bioresource Project
(NBRP), Japan.
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Table S1. The sequences and its purposes of PCR
primers used in this study.
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