title: Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis.
abstract: The influence of different amino acid substitutions in the nitrilase from Pseudomonas fluorescens EBC191 (NitA) on the catalytical activity and the ability to form amides was investigated. The enzyme variant Glu137Ala was constructed because glutamate residues homologous to Glu137 are highly conserved among different members of the nitrilase superfamily and it has been suggested that these residues are indispensable for the hydrolysis of amides by enzymes belonging to the nitrilase superfamily. The enzyme variant Glu137Ala demonstrated less than 1 % of the wild-type activity but was still enzymatically competent to convert mandelonitrile to mandelic acid and mandeloamide. The tryptophan residue at position 188, which was previously identified as important for the amide forming capacity of the nitrilase, was exchanged by saturation mutagenesis for all other proteinogenic amino acids. Surprisingly, 18 of these 19 exchanges resulted in an increased formation of mandeloamide from (R,S)-mandelonitrile and three of these variants converted (R,S)-mandelonitrile to more than 90 % of mandeloamide. Furthermore, these modifications also resulted in a reversal of stereoselectivity and these variants formed in contrast to the wild-type enzyme and almost all other known nitrilases preferentially (S)-mandelic acid. The synthetic potential of one of these variants was demonstrated by the construction of recombinant E. coli clones which simultaneously expressed the nitrilase variant and the (S)-hydroxynitrile lyase (oxynitrilase) from the cassava plant (Manihot esculenta). These "bienzymatic catalysts" converted benzaldehyde plus cyanide almost exclusively to (S)-mandeloamide and did not show any inhibition in the presence of cyanide in concentrations up to 200 mM.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Sosedov
authors.forename: Andreas
authors.initials: A
authors.lastname: Stolz
mesh_terms: D000097:Acetonitriles / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism; D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism; D001547:Benzaldehydes / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D008333:Mandelic Acids / Q000378:metabolism; D002365:Manihot / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000097:Acetonitriles; D000577:Amides; D001547:Benzaldehydes; D003486:Cyanides; D004269:DNA, Bacterial; D008333:Mandelic Acids; D018698:Glutamic Acid; C045516:mandelonitrile; D000619:Aminohydrolases; C571702:arylacetonitrilase, Pseudomonas fluorescens EBC191; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase; C037938:mandelic acid; C032175:benzaldehyde