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, 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.