APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY 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 Received: 18 August 2014 /Revised: 10 December 2014 /Accepted: 14 December 2014 /Published online: 31 December 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-ter- mini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previ- ous 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 delet- ed decreased in both activity and thermostability with increas- ing 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 en- zymes, 306GERDST311, was shown to be necessary for func- tionality and explains the inactivity of the previously de- scribed CynD stut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interac- tions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimer- ization 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 stut Δ302 or enhance the activity of full-length CynD stut and therefore does not act as a general stability motif. Keywords Cyanide dihydratase . Nitrilase . Cyanide . Bioremediation Introduction The cyanide dihydratases are enzymes belonging to the nitrilase branch of the nitrilase superfamily (Pace and Brenner 2001). Nitrilases hydrolyze organic nitriles to carboxylic acids and ammonia under mild acidic or basic reaction conditions (Thimann and Mahadevan 1964), in this instance, cyanide to formic acid (Meyers et al. 1993). Although there are no crystal structures of members for any members of the nitrilase branch, there are structures of other members of the superfamily such as a DCase (N-carbamoyl-D-amino acid amidohydrolase) from Agrobacterium sp. strain KNK712 (Nakai et al. 2000), NitFhit (nit-fragile histidine triad fusion) protein from Caenorhabditis elegans (Pace et al. 2000), a putative cyanide hydrolase from Saccharomyces cerevisiae (Kumaran et al. 2003) as well as several amidases (Hung et al. 2007; Kimani et al. 2007). Models have been proposed for several members of the nitrilase branch (Sewell et al. 2003; Thuku et al. 2009; Thuku et al. 2007) based on three-dimensional electron mi- croscopy and the fitting of homology models obtained by alignment of sequences to related crystallographically deter- mined structures. Members of the superfamily all possess sim- ilar monomer and dimer structures (Pace and Brenner 2001). The monomers fold into an αββα structure and then pair across the A surface to form αββα–αββα dimers. In the nitrilases, these dimers act as the building blocks for the Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6335-x) contains supplementary material, which is available to authorized users. M. A.<N. Crum : J. M. Park : M. J. Benedik (*) Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA e-mail: benedik@tamu.edu A. E. Mulelu : B. T. Sewell Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa Appl Microbiol Biotechnol (2015) 99:3093–3102 DOI 10.1007/s00253-014-6335-x