Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191.

Different members of the nitrilase superfamily (D-carbamoylases, Nit-Fhit proteins, amidases, cyanide dihydratases and nitrilases) were compared by multiple sequence alignments and a long carboxy-terminal extension (about 50 amino acids) identified in all nitrilases and cyanide dihydratases which was not present in other members of the nitrilase superfamily. The function of this C-terminal part was experimentally analysed in the arylacetonitrilase of Pseudomonas fluorescens EBC191 by the construction of various deletion mutants, chimeric enzymes with other bacterial nitrilases and site-specific mutagenesis. The enzyme variants were tested with the substrates 2-phenylpropionitrile and mandelonitrile and compared regarding specific activities, degree of amide formation and enantioselectivity. The enzyme variants containing deletions up to 32 amino acids did not show significant differences in comparison with the wild-type enzyme. Deletion mutants with 47-67 amino acids missing generally demonstrated reduced enzyme activities, increased amounts of amide formation and increased proportions of the (R)-enantiomers of the amides and acids formed. Also certain exchanges of H296 in the C-terminal motif DpvGHY led to enzyme variants with a similar phenotype. Chimeric enzymes which contained up to 59 amino acids deriving from the nitrilases of Rhodococcus rhodochrous NCIMB11216 or Alcaligenes faecalis ATCC8750 were active and resembled, with respect to the enantioselectivity and degree of amide formation, the wild-type enzyme of P.fluorescens.