Phenylalanine ammonia-lyases: combining protein engineering and natural diversity

RB Tomoiagă, SD Tork, A Filip, LC Nagy… - Applied Microbiology and …, 2023 - Springer
Applied Microbiology and Biotechnology, 2023Springer
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 …
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
Graphical abstract
Springer
以上显示的是最相近的搜索结果。 查看全部搜索结果