Abstract
In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) provided tailored PALs active towards challenging, highly valuable di-substituted substrates, such as the l-DOPA precursor 3,4-dimethoxy-l-phenylalanine or the 3-bromo-4-methoxy-phenylalanine. The rational design approach and saturation mutagenesis strategy unveiled identical PcPAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of PcPAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V PcPAL variants, we imprinted these beneficial mutations to PALs of different origins. The variants of PALs from Arabidopsis thaliana (AtPAL) and Anabaena variabilis (AvPAL) showed higher catalytic efficiency than their PcPAL homologues. Further, the engineered PALs were also compared in terms of catalytic efficiency with a novel aromatic ammonia-lyase from Loktanella atrilutea (LaAAL), close relative of the metagenome-derived aromatic ammonia-lyase AL-11, reported recently to possess atypically high activity towards substrates with electron-donor aromatic substituents. Indeed, LaAAL outperformed the engineered Pc/At/AvPALs in the production of 3,4-dimethoxy-l-phenylalanine; however, in case of 3-bromo-4-methoxy derivatives it showed no activity, with computational results supporting the occurrence of steric hindrance. Transferring the unique array of selectivity modulator residues from LaAAL to the well-characterized PALs did not enhance their activity towards the targeted substrates. Moreover, applying the rational design strategy valid for these well-characterized PALs to LaAAL decreased its activity. These results suggest that distinct tailoring rationale is required for LaAAL/AL-11-like aromatic ammonia-lyases, which might represent a distinct PAL subclass, with natural reaction and substrate scope modified through evolutionary processes.
Key points
• PAL-activity for challenging substrates generated by protein engineering
• Rational/semi-rational protein engineering reveals constrained mutational variability
• Engineered PALs are outperformed by novel ALs of distinct catalytic site signature
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Data availability
The Uniprot/EMBL identifiers of all protein/nucleotide sequences used within the alignments and experimental work and the Protein Data Bank (PDB) IDs for the protein structures used within the computational part are described within the manuscript or the Supplementary Information, while the original data that support the findings of this study are included within the manuscript/supplementary information and/or are available from the corresponding author upon reasonable request.
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Funding
This work was financed by the Romanian Ministry of Education and Research, CNCS–UEFISCDI, project number PN-III-P1-1.1-TE-2019–2118, within PNCDI III.
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R.B.T. was responsible for the saturation mutagenesis experiments, preparation of whole-cell-biocatalysts, reaction monitoring by HPLC, protein purification and enzyme kinetics. S.D.T. was responsible for the mutagenesis, activity measurements of the rationally designed PALs, while A.F. contributed to primer design, sequencing interpretation and protein purification. L.C.N. was responsible for computational studies and graphical artwork. L.C.B. conceived the project, was responsible for funding, supervised all experiments, data and wrote the paper together with R.B.T. All authors reviewed the manuscript.
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Supplementary file1. Supplementary information of this paper consists of the mutagenesis protocol and tables of primers employed for site-specific and saturation mutagenesis, library screening conditions and the alignment of PAL/TAL/HAL/AAL of distinct origins together with their Uniprot/EMBL identifiers. Information about functional characterization of purified enzymes is also provided, including HPLC monitoring conditions, conversion, and enantiomeric excess representative chromatograms, Michaelis–Menten curves and Tm values. Detailed description of the computational work is also included. (PDF 2.59 MB)
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Tomoiagă, R.B., Tork, S.D., Filip, A. et al. Phenylalanine ammonia-lyases: combining protein engineering and natural diversity. Appl Microbiol Biotechnol 107, 1243–1256 (2023). https://doi.org/10.1007/s00253-023-12374-x
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DOI: https://doi.org/10.1007/s00253-023-12374-x