Abstract
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 Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL 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, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, 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-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).
Highlights
Phenylalanine ammonia-lyases (PALs, EC 4.3.1.24, and PAL/TALs with combined phenylalanine and tyrosine ammonia-lyase activities, EC 4.3.1.25) play essential roles in the non-oxidative ammonia elimination of phenylalanine to cinnamic acid, an important precursor for the biosynthesis of phenylpropanoids
PALs from Rhodotorula glutinis (RgPAL) [1,2], Petroselinum crispum (PcPAL) [3], and Anabaena variabilis (AvPAL) [4] due to their broad substrate tolerance emerged as efficient biocatalysts for the production of phenylalanine analogs [5,6,7,8,9,10]
Among the PAL-activity assays, the high-performance liquid chromatography (HPLC) based procedure [14,16] allows the determination of conversion and enantiomeric excess values but can only be employed for the screening of limited sized libraries derived from rational design mutagenesis or single combinatorial active-site testing (CAST) [25]
Summary
Phenylalanine ammonia-lyases (PALs, EC 4.3.1.24, and PAL/TALs with combined phenylalanine and tyrosine ammonia-lyase activities, EC 4.3.1.25) play essential roles in the non-oxidative ammonia elimination of phenylalanine to cinnamic acid, an important precursor for the biosynthesis of phenylpropanoids. The reported fluorescence PAL-activity assays, despite their sensitivity, are limited for activity tests using the non-natural substrate o-NH2-phenylalanine [28] or require high instrumentation facilities, such as inductively coupled plasma–mass spectrometry (ICP-MS) [29], their general applicability is suppressed Another important issue is the quality of the transformant library, obtained through different mutagenesis methods, high-quality libraries, with a higher frequency of hits, providing the advantage of a reduced screening effort. The synthetic utility of I460V variant was demonstrated by the production of several m-, or p-substituted phenylalanine analogs of high synthetic value, employing biotransformations catalyzed efficiently by mutant I460V, but with low efficiency by wild-type PcPAL [18] In this context, this study focused on the randomization by site-saturation mutagenesis of the key activity modulator residue I460 of PcPAL in order to identify novel mutant variants with increased activity towards selected substrates of synthetic interest. For obtaining high-quality libraries, with increased frequency of hits within the transformant library, the mutagenesis procedure was optimized, while the PAL-activity assay was adapted to allow the high-throughput activity screens of cell lysates providing candidates of which superior activity was validated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
