Kataoka Shingo, Arakawa Takatoshi, Hori Shota, Katayama Yoko, Hara Yoshiko, Matsushita Yasuhiko, Nakayama Hiroshi, Yohda Masafumi, Nyunoya Hiroshi, Dohmae Naoshi, Maeda Mizuo, Odaka Masafumi
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.
FEBS Lett. 2006 Aug 21;580(19):4667-72. doi: 10.1016/j.febslet.2006.07.051. Epub 2006 Jul 24.
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.
硫氰酸盐水解酶（SCNase）是一种含钴酶，其半胱氨酸配体γCys131 - SO(2)H经过翻译后修饰。当SCNase的α、β和γ亚基在大肠杆菌中表达时，这些亚基组装形成异十二聚体(alphabetagamma)(4)，与天然SCNase相似，但不表现出催化活性。金属分析表明，无论培养基中是否存在钴，SCNase均以脱辅基形式表达。相反，在优化条件下，SCNase与位于SCNase基因下游编码的P15K在富含钴的培养基中共表达（SCNase((+P15K))），每个alphabetagamma三聚体含有0.86个钴原子，并表现出天然SCNase活性的78%。SCNase((+P15K))显示出SCNase钴中心特有的紫外可见吸收峰。约70%的SCNase((+P15K))具有γCys131 - SO(2)H修饰。这些结果表明SCNase((+P15K))是有活性的全酶形式的SCNase。P15K可能通过协助钴离子的掺入来促进SCNase的功能表达。
