Applied Microbiology and Biotechnology

, Volume 99, Issue 7, pp 3093–3102 | Cite as

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

  • Mary Abou-Nader Crum
  • Jason M. Park
  • Andani E. Mulelu
  • B. Trevor Sewell
  • Michael J. Benedik
Applied genetics and molecular biotechnology


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 (CynDpum) and P. stutzeri (CynDstut) 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, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-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 CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.


Cyanide dihydratase Nitrilase Cyanide Bioremediation 



The financial supports of The Welch Foundation (A1310) and the Texas Hazardous Waste Research Center are gratefully acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2014_6335_MOESM1_ESM.pdf (166 kb)
ESM 1 (PDF 166 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mary Abou-Nader Crum
    • 1
  • Jason M. Park
    • 1
  • Andani E. Mulelu
    • 2
  • B. Trevor Sewell
    • 2
  • Michael J. Benedik
    • 1
  1. 1.Department of BiologyTexas A&M UniversityCollege StationUSA
  2. 2.Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownCape TownSouth Africa

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