Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynD_pum) and P. stutzeri (CynD_stut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynD_stut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynD_stut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynD_stut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195–206 (A2) from CynD_pum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynD_stutΔ302 or enhance the activity of full-length CynD_stut and therefore does not act as a general stability motif.