Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties
Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of <i>Petroselinum crispum</i>...
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| Format: | Article |
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MDPI AG
2020-05-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/10/6/838 |
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| author | Raluca Bianca Tomoiagă Souad Diana Tork Ilka Horváth Alina Filip Levente Csaba Nagy László Csaba Bencze |
| author_facet | Raluca Bianca Tomoiagă Souad Diana Tork Ilka Horváth Alina Filip Levente Csaba Nagy László Csaba Bencze |
| author_sort | Raluca Bianca Tomoiagă |
| collection | DOAJ |
| description | Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of <i>Petroselinum crispum</i> PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify <i>Pc</i>PAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V <i>Pc</i>PAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, <i>p</i>-MeO-phenylalanine. Among the hits, besides the known I460V <i>Pc</i>PAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-<i>Pc</i>PAL in terms of catalytic efficiency within the reaction of <i>p</i>-MeO-Phe. Moreover, I460T <i>Pc</i>PAL maintained the high specificity constant of the <i>wild-type</i> enzyme for the natural substrate, <span style="font-variant: small-caps;">l</span>-Phe. Molecular docking supported the favorable substrate orientation of <i>p</i>-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V <i>Pc</i>PAL (PDB ID: 6RGS). |
| first_indexed | 2024-03-10T19:29:03Z |
| format | Article |
| id | doaj.art-c32d4e32defd493da21a30ab83933775 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T19:29:03Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-c32d4e32defd493da21a30ab839337752023-11-20T02:17:18ZengMDPI AGBiomolecules2218-273X2020-05-0110683810.3390/biom10060838Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic PropertiesRaluca Bianca Tomoiagă0Souad Diana Tork1Ilka Horváth2Alina Filip3Levente Csaba Nagy4László Csaba Bencze5Biocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, RomaniaPhenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of <i>Petroselinum crispum</i> PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify <i>Pc</i>PAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V <i>Pc</i>PAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, <i>p</i>-MeO-phenylalanine. Among the hits, besides the known I460V <i>Pc</i>PAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-<i>Pc</i>PAL in terms of catalytic efficiency within the reaction of <i>p</i>-MeO-Phe. Moreover, I460T <i>Pc</i>PAL maintained the high specificity constant of the <i>wild-type</i> enzyme for the natural substrate, <span style="font-variant: small-caps;">l</span>-Phe. Molecular docking supported the favorable substrate orientation of <i>p</i>-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V <i>Pc</i>PAL (PDB ID: 6RGS).https://www.mdpi.com/2218-273X/10/6/838biocatalysisphenylalanine ammonia-lyasessaturation mutagenesisprotein engineering |
| spellingShingle | Raluca Bianca Tomoiagă Souad Diana Tork Ilka Horváth Alina Filip Levente Csaba Nagy László Csaba Bencze Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties Biomolecules biocatalysis phenylalanine ammonia-lyases saturation mutagenesis protein engineering |
| title | Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties |
| title_full | Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties |
| title_fullStr | Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties |
| title_full_unstemmed | Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties |
| title_short | Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties |
| title_sort | saturation mutagenesis for phenylalanine ammonia lyases of enhanced catalytic properties |
| topic | biocatalysis phenylalanine ammonia-lyases saturation mutagenesis protein engineering |
| url | https://www.mdpi.com/2218-273X/10/6/838 |
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