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Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

ABSTRACT: The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.

SUBMITTER: Kiziak C

PROVIDER: S-EPMC2737917 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

Kiziak Christoph C Stolz Andreas A

Applied and environmental microbiology 20090706 17

The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC ...[more]

PMID: 19581475

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Project description:The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.

Dataset Information

Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

Publications

Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

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