Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K

Abstract

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, cCys131-SO 2 H. When the SCNase a, b and c subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (abc) 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/abc 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 cCys131-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.

Discovery of posttranslational maturation by self-subunit swapping

“…Metal ion binding is responsible for the posttranslational modification of the two cysteines in the ␣-subunit in NHase (1,12), but such modifications cannot be observed in apo-NHase or cobalt-free SCNase (12,29). These results permit us to speculate that the cysteine residues are modified in holo-␣e 2 , but not in apo-␣e 2 (purified from the culture in the absence of cobalt) ( Table 1).…”

“…1 C and D (4,28)] of L-NHase derived from nhlBAE was found to be 0.88 Ϯ 0.04 mol/mol of ␣␤, whereas those of the heterodimer and heterotetramer derived from nhlBA were very low (Table 1). Whereas the active enzyme is presumed to contain the two oxidized cysteine ligands (8,9,13,14), the apoprotein is likely to be nonmodified, judging from previous studies of NHase (12) and the related enzyme thiocyanate hydrolase (SCNase) (29). These findings suggest that the gene products derived from nhlBA are apo-L-NHases (apo-␣␤ and apo-␣ 2 ␤ 2 , respectively), in contrast to the holo-L-NHase (holo-␣ 2 ␤ 2 ) derived from nhlBAE.…”

Structure of Thiocyanate Hydrolase: A New Nitrile Hydratase Family Protein with a Novel Five-coordinate Cobalt(III) Center

“…chromatography-multi-angle light scattering measurement. 22 Four αβγ hetero-trimers (T1, T2, T3 and T4) are arranged with non-crystallographic pseudo-222 point group symmetry. An αβγ hetero-trimer is superimposable to the others with an RMSD value of less than 0.146 Å and 0.159 Å for the all C α atoms in native and apo-SCNase, respectively (Table 1).…”

“…1 Very recently, we have developed a recombinant expression system of SCNase in Escherichia coli. 22 When only the coding genes were expressed in E. coli, SCNase was expressed as an apo-enzyme that exhibited no SCNase activity. No post-translational modification was detected by mass spectrometric measurement.…”

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201

“…thioparus THI115 contains a non-corrin cobalt active centre and has two post-translationally modified cysteine ligands, i.e. cysteine sulfenic acid (or cysteine sulfenate) and cysteine sulfininate; the enzyme requires co-expression of an activator protein, P15K, for its functional expression (Kataoka et al, 2006;Katayama et al, 2006;Arakawa et al, 2007). The presence of a similar enzyme in the halophilic chemolithoautotroph Thiohalophilus (Th.)…”