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Applied and Environmental Microbiology, September 2009, p. 5592-5599, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00301-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191 ^,

Christoph Kiziak and Andreas Stolz^*

Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

Received 6 February 2009/ Accepted 27 June 2009

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.

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* Corresponding author. Mailing address: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49 (0)711 68565489. Fax: 49 (0)711 68565725. E-mail: andreas.stolz{at}imb.uni-stuttgart.de

Published ahead of print on 6 July 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Lonza AG, LBPSi Microbial Research and Development, CH-3930 Visp, Switzerland.

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Applied and Environmental Microbiology, September 2009, p. 5592-5599, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00301-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.