caa a22 4500 605500223 CHVBK 20210128100554.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6335-x doi (NATIONALLICENCE)springer-10.1007/s00253-014-6335-x Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein [Elektronische Daten] [Mary Crum, Jason Park, Andani Mulelu, B. Sewell, Michael Benedik] 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. Springer-Verlag Berlin Heidelberg, 2014 Cyanide dihydratase nationallicence Nitrilase nationallicence Cyanide nationallicence Bioremediation nationallicence Crum Mary Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut Park Jason Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut Mulelu Andani Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa aut Sewell B. Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa aut Benedik Michael Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/7(2015-04-01), 3093-3102 0175-7598 99:7<3093 2015 99 253 https://doi.org/10.1007/s00253-014-6335-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6335-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Crum Mary Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut NATIONALLICENCE 700 1- Park Jason Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut NATIONALLICENCE 700 1- Mulelu Andani Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa aut NATIONALLICENCE 700 1- Sewell B. Structural Biology Research Unit, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa aut NATIONALLICENCE 700 1- Benedik Michael Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/7(2015-04-01), 3093-3102 0175-7598 99:7<3093 2015 99 253