TY - JOUR
T1 - Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K
AU - Kataoka, Shingo
AU - Arakawa, Takatoshi
AU - Hori, Shota
AU - Katayama, Yoko
AU - Hara, Yoshiko
AU - Matsushita, Yasuhiko
AU - Nakayama, Hiroshi
AU - Yohda, Masafumi
AU - Nyunoya, Hiroshi
AU - Dohmae, Naoshi
AU - Maeda, Mizuo
AU - Odaka, Masafumi
N1 - Funding Information:
We thank Prof. M. Horio, Dr. R. Noda and A. Honya of Tokyo University of Agriculture and Technology for the analysis of Co concentration. We thank T. Fujii and M. Kuramoto for technical assistance. The work reported here is a part of the 21st Century COE (Center of Excellence) program of “Future Nano-Materials” research and education project, which is financially supported by the Ministry of Education, Science, Sports, Culture, and Technology through Tokyo University of Agriculture & Technology. This work was supported by the Bioarchitect Research program of RIKEN (to M.O.), a grant-in-aid for scientific research (B) (12440191), grants-in-aid for scientific research on priority areas (15032212, 17028013), and a grant of the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports and Culture of Japan (to M.Y.).
PY - 2006/8/21
Y1 - 2006/8/21
N2 - Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)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/αβγ 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 γCys131-SO2H 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.
AB - Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)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/αβγ 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 γCys131-SO2H 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.
KW - Activator protein
KW - Apo-protein
KW - Cysteine-sulfenic acid
KW - Cysteine-sulfinic acid
KW - Nitrile hydratase
KW - Non-corrin cobalt
KW - Post-translational modification
KW - Thiocyanate hydrolase
UR - http://www.scopus.com/inward/record.url?scp=33746918762&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33746918762&partnerID=8YFLogxK
U2 - 10.1016/j.febslet.2006.07.051
DO - 10.1016/j.febslet.2006.07.051
M3 - Article
C2 - 16879822
AN - SCOPUS:33746918762
SN - 0014-5793
VL - 580
SP - 4667
EP - 4672
JO - FEBS Letters
JF - FEBS Letters
IS - 19
ER -