Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K
Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K
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DOI：
10.1016/j.febslet.2006.07.051
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发表时间：
2006-08-21
期刊：
影响因子：
3.5
通讯作者：
Odaka, Masafumi
中科院分区：
文献类型：
--
作者：
Kataoka, Shingo;Arakawa, Takatoshi;Odaka, Masafumi
硫氰酸盐水解酶(SCNase)是一种含钴的酶，具有半胱氨酸的翻译后修饰配体--γ-Cys131-SO2H。当SCNaseα、β和伽马亚基在大肠杆菌中表达时，这些亚基组装成一个杂十二聚体(αβ伽马)(4)，与天然的SCNase相似，但没有催化活性。金属分析表明，SCNase以脱氧核糖核酸酶的形式表达，与介质中钴的存在无关。相反，在优化条件下(SCNase((+Pi5K)与编码在SCNase基因下游的P15K共表达的SCNase具有0.86Co原子/α-β-γ三聚体，其活性是天然SCNase的78%。SCNase((+Pi5K))具有SCNase钴中心的紫外-可见吸收峰特征。约70%的SCNase(+P15K)具有γ-Cys131-SO2H修饰。这些结果表明，SCNase((+P15K))是具有活性的完整SCNase。P15K可能通过辅助钴离子的掺入而促进SCNase的功能表达。(C)2006年欧洲生化学会联合会。爱思唯尔出版，版权所有。
Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gamma Cys131-SO2H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alpha beta gamma)(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((+Pi5K))) possessed 0.86 Co atom/alpha beta gamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+Pi5K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gamma Cys131-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. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.