Purification, kinetic characterization, and molecular cloning of a novel enzyme, ecdysteroid 22-kinase.
This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365-26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase.
Sonobe
Haruyuki
H
Department of Biology, Faculty of Science and Engineering, Konan University, Kobe 658-8501, Japan. sonobe@konan-u.ac.jp
Ohira
Tsuyoshi
T
Ieki
Katsunori
K
Maeda
Sayaka
S
Ito
Yoichi
Y
Ajimura
Masahiro
M
Mita
Kazuei
K
Matsumoto
Hitoshi
H
Wilder
Marcy N
MN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology; D003001:Cloning, Molecular; D026461:Ecdysteroids / Q000096:biosynthesis*; D005260:Female; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology; D010063:Ovum / Q000201:enzymology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D026461:Ecdysteroids; D019476:Insect Proteins; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C514949:ecdysteroid 22-kinase, Bombyx mori