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

Abstract

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.

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

“…Multiple-sequence alignments demonstrated that the corresponding tyrosine residue is highly conserved among different nitrilases and amidases from bacterial, plant, or fungal sources (21,45). The importance of this residue for the catalytic activity of nitrilases was experimentally verified during our studies, as several of the enzyme variants generated (e.g., Tyr54Ile, Tyr54Lys, and Tyr54Arg) were completely inactive for the conversion of acetophenone cyanohydrin, mandelonitrile, and 2-phenylpropionitrile.…”

“…The construction of plasmids pIK7, pIK9, and pDHE22, which encode the nitrilases from Alcaligenes faecalis ATCC 8750, Pseudomonas fluorescens EBC191, and Synechocystis sp. strain PCC6803, respectively, was described previously (16,20,21). The gene for the rhodococcal nitrilase was amplified from a nitrileconverting Rhodococcus rhodochrous strain.…”

“…DNA preparation, DNA manipulation, transformation, PCR, DNA sequencing, database searches, and sequence alignments were performed as described previously (21). Plasmid DNA was custom sequenced by GATC Biotech (Konstanz, Germany).…”

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

“…This enzyme forms with certain nitriles also significant amounts of the corresponding amides as side products (3,5,8,15,19,24). The enzyme has recently been studied intensively in order to analyze the molecular basis for the substrate specificity, reaction specificity, and enantiospecificity of nitrilases (9,10). In the course of these investigations, the effects of various carboxy-terminal mutations and mutations in close proximity to the catalytic active cysteine residue were analyzed.…”

“…In the course of these investigations, the effects of various carboxy-terminal mutations and mutations in close proximity to the catalytic active cysteine residue were analyzed. These experiments demonstrated that deletions of 47 to 67 amino acids (aa) from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated increased amide formation and an increased formation of the (R)-acids (9). In addition, it was demonstrated that the size of the amino acid residue in direct proximity to the catalytic active cysteine residue (toward the C terminus) is determinative of the enantioselectivity of acid formation.…”