title: Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein.
abstract: 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.
authors.affiliation: Department of Biology, Texas A&M University, College Station, TX, 77843-3258, USA.
authors.forename: Mary Abou-Nader
authors.initials: MA
authors.lastname: Crum
authors.forename: Jason M
authors.initials: JM
authors.lastname: Park
authors.forename: Andani E
authors.initials: AE
authors.lastname: Mulelu
authors.forename: B Trevor
authors.initials: BT
authors.lastname: Sewell
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Benedik
mesh_terms: D000409:Alanine; D000619:Aminohydrolases / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology; D055503:Protein Multimerization; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000378:metabolism
chemical_list: D006867:Hydrolases; D000619:Aminohydrolases; C083488:cyanide dihydratase; C021867:nitrilase; D006836:Hydro-Lyases; C076848:cyanide hydratase; D000409:Alanine