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 (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.

Particularities of Telework Applicable to the Health System in the Context of the COVID-19 Pandemic.

The paper aims to highlight how physician-patient relationships have evolved amid the COVID-19 pandemic by considering telework implementation into the healthcare sector. The article presents the peculiarities of using telework within the medical system given the recent epidemiological context, by pointing out the advantages and disadvantages of its adoption. To achieve this goal, a qualitative marketing research was conducted to identify physicians' opinions and perceptions on telework. The main objectives were identifying the ways of using telework, the effects that telework has on the quality of the medical services and on patient relationships, as well as the strengths and weaknesses of telework for the medical field. The study revealed that while face-to-face consultations decreased as the outbreak continued, different methods of remote consultations emerged, which was both beneficial in interactions with chronic patients and detrimental, as medical staff became more and more overworked. For these reasons, our research shows that healthcare professionals consider a hybrid system much more adequate for patients with stable chronic conditions, as ongoing monitoring is done through this remote mechanism.

Towards a general approach for tailoring the hydrophobic binding site of phenylalanine ammonia-lyases.

Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL). With some exceptions, the results support that the positions of substrate specificity modulating residues are conserved among PALs, thus the mutation with beneficial effect for PAL-activity enhancement can be predicted using the established rational design model. Accordingly, the study supports that tailoring PALs of different origins and different substrate scope, can be performed through a general method. Moreover, the fact that AtPAL variants I461V, L133A and L257V, all outperformed in terms of catalytic efficiency the corresponding, previously reported, highly efficient PcPAL variants, of identical catalytic site, suggests that not only catalytic site differences influence the PAL-activity, thus for the selection of the optimal PAL-biocatalysts for a targeted process, screening of PALs from different origins, should be included.

Toolbox for the structure-guided evolution of ferulic acid decarboxylase (FDC).

The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

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 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).

Exploring the substrate scope of ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae.

Ferulic acid decarboxylase from Saccharomyces cerevisiae (ScFDC1) was described to possess a novel, prenylated flavin mononucleotide cofactor (prFMN) providing the first enzymatic 1,3-dipolar cycloaddition mechanism. The high tolerance of the enzyme towards several non-natural substrates, combined with its high quality, atomic resolution structure nominates FDC1 an ideal candidate as flexible biocatalyst for decarboxylation reactions leading to synthetically valuable styrenes. Herein the substrate scope of ScFDC1 is explored on substituted cinnamic acids bearing different functional groups (-OCH3, -CF3 or -Br) at all positions of the phenyl ring (o-, m-, p-), as well as on several biaryl and heteroaryl cinnamic acid analogues or derivatives with extended alkyl chain. It was found that E. coli whole cells expressing recombinant ScFDC1 could transform a large variety of substrates with high conversion, including several bulky aryl and heteroaryl cinnamic acid analogues, that characterize ScFDC1 as versatile and highly efficient biocatalyst. Computational studies revealed energetically favoured inactive binding positions and limited active site accessibility for bulky and non-linear substrates, such as 2-phenylthiazol-4-yl-, phenothiazine-2-yl- and 5-(4-bromophenyl)furan-2-yl) acrylic acids. In accordance with the computational predictions, site-directed mutagenesis of residue I330 provided variants with catalytic activity towards phenothiazine-2-yl acrylic acid and provides a basis for altering the substrate specificity of ScFDC1 by structure based rational design.

Tailored Mutants of Phenylalanine Ammonia-Lyase from Petroselinum crispum for the Synthesis of Bulky l- and d-Arylalanines.

Tailored mutants of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)-arylacrylates. The wild-type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well-studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1'-biphenyl]-4-yl)-, (4'-fluoro-[1,1'-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.

Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.