Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K.
Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.
Kataoka
Shingo
S
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Arakawa
Takatoshi
T
Hori
Shota
S
Katayama
Yoko
Y
Hara
Yoshiko
Y
Matsushita
Yasuhiko
Y
Nakayama
Hiroshi
H
Yohda
Masafumi
M
Nyunoya
Hiroshi
H
Dohmae
Naoshi
N
Maeda
Mizuo
M
Odaka
Masafumi
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D017931:DNA Primers; D006867:Hydrolases; C074369:thiocyanate hydrolase