Tài liệu Báo cáo khoa học: Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of

Molecular defect of isovaleryl-CoA dehydrogenase in the

skunk mutant of silkworm, Bombyx mori

Kei Urano1

, Takaaki Daimon1

, Yutaka Banno2

, Kazuei Mita3

, Tohru Terada4

, Kentaro Shimizu4,5,

Susumu Katsuma1 and Toru Shimada1,4

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

sequences and employ a similar enzyme mechanism for

the a,b-dehydrogenation of acyl-CoA substrates [1].

Keywords

Bombyx mori; branched-chain amino acid;

isovaleric acidemia; isovaleryl-CoA

dehydrogenase; responsible gene

Correspondence

T. Shimada, Laboratory of Insect Genetics

and Bioscience, Department of Agricultural

and Environmental Biology, Graduate School

of Agricultural and Life Sciences, University

of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo

113-8657, Japan

Fax: +81 3 5841 8011

Tel: +81 3 5841 8124

E-mail: [email protected]

(Received 2 March 2010, revised 1 August

2010, accepted 25 August 2010)

doi:10.1111/j.1742-4658.2010.07832.x

The isovaleric acid-emanating silkworm mutant skunk (sku) was first stud￾ied over 30 years ago because of its unusual odour and prepupal lethality.

Here, we report the identification and characterization of the gene responsi￾ble for the sku mutant. Because of its specific features and symptoms simi￾lar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known

as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be

responsible for the phenotype of the sku mutant. Linkage analysis revealed

that the silkworm IVD gene (BmIVD) was closely linked to the odorous

phenotype as expected, and a single amino acid substitution (G376V) was

found in BmIVD of the sku mutant. To investigate the effect of the G376V

substitution on BmIVD function, wild-type and sku-type recombinants

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