Phenylalanine ammonia-lyases: combining protein engineering and natural diversity

In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum ( Pc PAL) 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 Pc PAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of Pc PAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V Pc PAL variants, we imprinted these beneficial mutations to PALs of different origins. The variants of PALs from Arabidopsis thaliana ( At PAL) and Anabaena variabilis ( Av PAL) showed higher catalytic efficiency than their Pc PAL homologues. Further, the engineered PALs were also compared in terms of catalytic efficiency with a novel aromatic ammonia-lyase from Loktanella atrilutea ( La AAL), 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, La AAL outperformed the engineered Pc / At / Av PALs 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 La AAL 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 La AAL decreased its activity. These results suggest that distinct tailoring rationale is required for La AAL/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 Graphical abstract